IL10Rb binding molecule and method of use

Abstract

This invention provides a polypeptide that specifically binds to the extracellular domain of IL10Rb. [Solution] The present invention provides a biologically active molecule containing a single-domain antibody (sdAb) that specifically binds to the extracellular domain of human IL10Rb, a composition containing such an antibody, and a method of using the same.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 061,562, filed Aug. 5, 2020; U.S. Provisional Application No. 63 / 078,745, filed Sep. 15, 2020; and U.S. Provisional Application No. 63 / 135,884, filed Jan. 11, 2021. The disclosures of these provisional applications are hereby incorporated by reference in their entirety for all purposes.

[0002] Field of the Invention The present disclosure relates to biologically active molecules comprising single - domain antibodies that specifically bind to the extracellular domain of IL10Rb, compositions comprising such single - domain antibodies, and methods of using the same.

Background Art

[0003] Background The anti - inflammatory cytokine interleukin - 10 (IL - 10) is also known as human cytokine synthesis inhibitory factor (CSIF) and is classified as a type (class) - 2 cytokine, a set of cytokines that includes IL - 19, IL - 20, IL - 22, IL - 24 (Mda - 7), and IL - 26, interferons (IFN - α, - β, - γ, - δ, - ε, - κ, - Ω, and - τ) and interferon - like molecules (limitin, IL - 28A, IL - 28B, and IL - 29). Human IL - 10 is a homodimer with a molecular weight of 37 kDa, and each 18.5 kDa monomer contains 178 amino acids, the first 18 of which include a signal peptide and two cysteine residues that form two intramolecular disulfide bonds.

[0004] The IL-10 receptor, a type II cytokine receptor, consists of alpha and beta subunits, also referred to as R1 and R2, respectively. Receptor activation requires binding to both alpha and beta. One homodimer of the IL-10 polypeptide binds to alpha, while the other homodimer of the same IL-10 polypeptide binds to beta. In addition to forming a subunit of the ILRb receptor complex, the IL10Rb receptor subunit is a component of the IL22, IL26, IL28, and interferon-lambda L1 receptor complex, the IFNL1 variant. The IFNL1 / IL10RB dimer is a receptor for the cytokine ligands IFNL2 and IFNL3, mediating their antiviral activity. IL10Rb is also known as CDW210B. In contrast to IL10Ra, which is primarily expressed on hematopoietic cells, the IL10Rb receptor subunit is ubiquitous. The interaction between IL-10 and IL-10Ra is a specific high-affinity interaction, while the association of IL-10 with IL-10Rb is a low-affinity shared receptor. Reports suggest that the interaction of IL-10 with IL-10Ra induces conformational changes in IL-10Rb, thereby promoting its binding to IL-10.

[0005] Human IL10Rb (hIL10Rb) is expressed as a 325-amino acid preprotein containing a 19-amino acid N-terminal signal sequence. Amino acids 20-220 (amino acids 1-201 in the mature protein) correspond to the extracellular domain, amino acids 221-242 (amino acids 202-223 in the mature protein) correspond to the 22-amino acid transmembrane domain, and amino acids 243-325 (amino acids 224-306 in the mature protein) correspond to the intracellular domain. hIL10Rb is referenced as entry Q08334 in the UniProtKB database. Mouse IL10Rb (mIL10Rb) is expressed as a 349-amino acid preprotein containing a 19-amino acid N-terminal signal sequence. Amino acids 20-220 (amino acids 1-201 in mature proteins) correspond to the extracellular domain, amino acids 221-241 (amino acids 202-222 in mature proteins) correspond to the 21-amino acid transmembrane domain, and amino acids 242-349 (amino acids 223-330 in mature proteins) correspond to the intracellular domain. mCD132 is referenced as entry Q61190 in the UniProtKB database.

[0006] IL-10 exerts multifaceted effects in immunomodulation and inflammation through its actions on T cells, B cells, macrophages, and antigen-presenting cells (APCs). IL-10 is produced by mast cells and counteracts the inflammatory effects these cells have at the site of allergic reactions. While primarily expressed in macrophages, IL-10 expression has also been detected in activated T cells, B cells, mast cells, and monocytes. IL-10 can suppress immune responses by inhibiting the expression of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF in activated monocytes and activated macrophages, and it also suppresses IFN-γ production by NK cells. IL-10 can block NF-κB activity and is involved in regulating the JAK-STAT signaling pathway.

[0007] Monoclonal antibodies are the most widely used reagents for protein detection and quantification; however, monoclonal antibodies are large molecules of approximately 150 kDa, which can sometimes limit their use in assays involving several competing reagents for recognition of closely spaced epitopes. A unique class of immunoglobulins containing a heavy chain domain and lacking a light chain domain (commonly called "heavy chain" antibodies (HCAb)) is found in camelids, including dromedaries, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicuñas, and guanacos, as well as cartilaginous fish such as sharks. The isolated variable domain region of HCAb is known as VHH (an abbreviation for "variable-heavy-heavy," reflecting its structure) or Nanobody® (Ablynx). Single-domain VHH antibodies have the advantage of being small in size (approximately 12-14 kD), about 1 / 10 the molecular weight of conventional mammalian IgG class antibodies. This facilitates the binding of these VHH molecules to target antigenic determinants that conventional monoclonal IgG forms may not be able to reach (Ingram et al., 2018). Furthermore, VHH single-domain antibodies often feature high thermal stability, which facilitates drug delivery to areas where cold chain infrastructure is difficult or impossible to establish. When these properties are combined, in particular, with simple phage display recovery methods that do not require heavy / light chain pair formation (as in the case of IgG antibodies) and simple manufacturing (e.g., manufacturing in bacterial expression systems), VHH single-domain antibodies are useful in a variety of applications, including the development of imaging and therapeutic agents. [Overview of the project]

[0008] This disclosure provides a polypeptide that specifically binds to the extracellular domain of IL10Rb.

[0009] This disclosure provides an IL10Rb binding molecule that specifically binds to the extracellular domain of IL10Rb (e.g., human or mouse IL10Rb).

[0010] In some embodiments, the IL10Rb-binding molecule includes a single-domain antibody (sdAb) that specifically binds to the extracellular domain of human IL10Rb (hIL10Rb).

[0011] In some embodiments, the IL10Rb-binding molecule is an sdAb, which comprises a set of CDRs corresponding to CDR1, CDR2, and CDR3, as shown in the horizontal rows of Table 1 below.

[0012] In some embodiments, the IL10Rb binding molecule comprises CDR1, CDR2, and CDR3 as shown in the rows of Table 1 below, and each of CDR1, CDR2, and CDR3 independently may have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the sequences shown in the rows of Table 1 below, and may have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

[0013] In some embodiments, the IL10Rb-binding molecule consists of, optionally essentially, or optionally includes, a single-domain antibody (sdAb) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity (or identical except for one, two, three, or four amino acids that are optionally conserved substitutions) or 100% identity with any one polypeptide sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, and 105, as shown in Table 1 below.

[0014] (Table 1) hIL10Rb VHH and CDR amino acid (AA) sequences TIFF2026086659000001.tif221167TIFF2026086659000002.tif221167TIFF2026086659000003.tif221167TIFF2026086659000004.tif191167

[0015] (Table 2) Nucleic acid sequences encoding hIL10Rb binding molecules TIFF2026086659000005.tif233167TIFF2026086659000006.tif233167TIFF2026086659000007.tif23316 7TIFF2026086659000008.tif233167TIFF2026086659000009.tif233167TIFF2026086659000010.tif98167

[0016] Mouse IL10Rb In some embodiments, IL10Rb is mouse IL10Rb.

[0017] In some embodiments, the IL10Rb-binding molecule comprises a single-domain antibody (sdAb) that specifically binds to the extracellular domain of mouse or rat IL10Rb (mIL10Rb).

[0018] In some embodiments, the IL10Rb-binding molecule is an sdAb, which comprises a set of CDRs corresponding to CDR1, CDR2, and CDR3, as shown in the horizontal rows of Table 3 below.

[0019] In some embodiments, the IL10Rb binding molecule comprises CDR1, CDR2, and CDR3 as shown in the rows of Table 3 below, and each of CDR1, CDR2, and CDR3 independently may have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the sequences shown in the rows of Table 3 below, and may have 0, 1, 2, or 3 amino acid changes, and optionally, conservative amino acid changes.

[0020] In some embodiments, the IL10Rb-binding molecule consists of, optionally essentially, or optionally includes a single-domain antibody (sdAb) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity (or optionally identical except for one, two, three, or four conservative substitutions) or 100% identity with respect to any one polypeptide sequence of SEQ ID NO: 136, 140, 144, 148, 152, and 156, as shown in Table 3 below.

[0021] (Table 3) mM10Rb VHH and CDR amino acid (AA) sequences TIFF2026086659000011.tif195167

[0022] Furthermore, this disclosure provides nucleic acids encoding an mRNA10Rb-binding molecule. Table 4 below shows an example of a DNA sequence encoding an mRNA10Rb-binding molecule, as described in Table 3 above.

[0023] (Table 4) DNA sequences encoding mRNA10RB VHH TIFF2026086659000012.tif217159

[0024] In some embodiments, mouse IL10Rb-binding molecules are useful as substitutes for human IL10Rb molecules for evaluating activity in mouse models.

[0025] This disclosure further provides recombinant viral and nonviral vectors comprising nucleic acids encoding the IL10Rb binding molecule of this disclosure or the CDR of the IL10Rb binding molecule of this disclosure.

[0026] This disclosure further provides host cells comprising recombinant viruses and nonviral vectors containing the nucleic acid of the IL10Rb binding molecule of this disclosure or the CDR of the IL10Rb binding molecule of this disclosure.

[0027] This disclosure further provides host cells comprising recombinant viruses and nonviral vectors containing the nucleic acid of the IL10Rb binding molecule of this disclosure or the CDR of the IL10Rb binding molecule of this disclosure.

[0028] This disclosure further provides pharmaceutical formulations including recombinant viruses and nonviral vectors containing the nucleic acid of the IL10Rb binding molecule of this disclosure, as well as methods of using them in the treatment or prevention of diseases, disorders, or conditions in mammalian subjects.

[0029] This disclosure further includes a kit comprising the IL10Rb binding molecule of this disclosure.

[0030] In another aspect, the Disclosure provides constructs for the targeted delivery of therapeutic agents to cells expressing the IL10Rb receptor, wherein an IL10Rb-binding molecule is conjugated to one or more therapeutic agents, optionally chemically or via a polypeptide linker. The Disclosure further provides a method of the above use in the treatment of a disease associated with IL10Rb expression in a subject, comprising administering a therapeutically effective amount of the IL10Rb-binding molecule conjugated to a subject requiring treatment, either alone or in combination with one or more additional therapeutic agents. In some embodiments, the diseases suitable for treatment are diseases, disorders, or conditions associated with signaling from receptors containing IL10Rb. In some embodiments, the IL10Rb-binding molecule of the Disclosure is useful in the treatment of diseases associated with dysregulated T-cell or B-cell activity.

[0031] In another aspect, the Disclosure provides constructs for identifying IL10Rb receptor-expressing cells, wherein an IL10Rb-binding molecule is optionally conjugated to one or more imaging agents via a chemical linker or polypeptide linker. Furthermore, the Disclosure provides a method for the aforementioned use in identifying IL10Rb receptor-expressing cells in a subject, comprising the steps of administering an effective amount of an IL10Rb-binding molecule conjugated to an imaging agent to a subject requiring treatment, and evaluating the subject for the presence of the imaging agent conjugated to the IL10Rb-binding molecule.

[0032] In another aspect, the present disclosure provides an IL10Rb-binding molecule modified to extend the duration of action in vivo, wherein the IL10Rb-binding molecule is conjugated to one or more carrier molecules.

[0033] This disclosure provides an IL10Rb-binding molecule comprising a polypeptide sequence that specifically binds to the extracellular domain of IL10Rb, and a method for using the same in the isolation, depletion, or enrichment of IL10Rb-expressing cells in a biological sample. [Invention 1001] An IL10Rb-binding molecule that specifically binds to the extracellular domain of IL10Rb. [Invention 1002] An IL10Rb-binding molecule according to the present invention 1001, containing a single-domain antibody (sdAb). [Invention 1003] sdAb is shown in the following table: The IL10Rb-binding molecule of the present invention 1002, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3, as shown in the row of TIFF2026086659000013.tif126164. [Invention 1004] An IL10Rb-binding molecule of the present invention 1002 or 1003, wherein sdAb has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one polypeptide sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, and 105. [Invention 1005] sdAb is shown in the following table: The IL10Rb-binding molecule of the present invention 1002, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3, as shown in the row of TIFF2026086659000014.tif34167. [Invention 1006] An IL10Rb-binding molecule of the present invention 1002, wherein sdAb has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one polypeptide sequence of SEQ ID NO: 136, 144, 148, 152, and 156. [Invention 1007] An IL10Rb-binding molecule according to either Invention 1003 or 1005, comprising a humanized or otherwise CDR grafted onto a heterogeneous framework. [Invention 1008] An IL10Rb-binding molecule according to any one of the present invention 1001 to 1007, further comprising a labeling agent, an imaging agent, and / or a therapeutic agent. [Invention 1009] A method for treating or preventing a disease, disorder, or condition in a mammalian subject by administering a therapeutically effective amount of any IL10Rb-binding molecule or a pharmaceutically acceptable formulation thereof of any of the invention items 1001 to 1008 to the mammalian subject. [Invention 1010] An IL10Rb-binding molecule according to any of invention 1001-1008 for use in the isolation, depletion, or enrichment of IL10Rb+ cells from a biological sample. [Invention 1011] A nucleic acid sequence encoding any of the IL10Rb binding molecules of the present invention 1001 to 1008. [Invention 1012] A recombinant virus or non-viral vector comprising the nucleic acid of the present invention 1011. [Invention 1013] A host cell containing the nucleic acid of the present invention 1011. [Invention 1014] A pharmaceutical formulation comprising the virus or nonviral vector of the present invention 1012. [Invention 1015] A kit comprising any IL10Rb-binding molecule according to invention 1001-1008. [Modes for carrying out the invention]

[0034] Detailed description of the invention Introduction To facilitate a more readily understandable understanding of this disclosure, certain terms and phrases are defined below and throughout this Spec. The definitions set forth herein are not limiting and should be interpreted in light of the knowledge of those skilled in the art.

[0035] Before describing the methods and compositions described herein, it should be understood that this disclosure is not limited to the specific methods or compositions described and, of course, may be modified.

[0036] Where a range of values ​​is defined, it is understood that each intermediate value between the upper and lower limits of that range is also disclosed in detail, down to 1 / 10 of the lower limit unit, unless otherwise explicitly specified by the context. Each narrow range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within that stated range is included in the present invention. The upper and lower limits of these narrow ranges may be independently included in or excluded from this range, and each range that includes one limit, does not include either limit, or includes both limits is also included in the present invention, depending on any limit that is explicitly limited and excluded within the stated range. Where the stated range includes one or both limits, a range that excludes either or both of the included limits is also included in the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but several possible and preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference to disclose and illustrate the methods and / or materials described in the cited publications.

[0038] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references unless specifically defined by the context. Thus, for example, a reference to “one cell” includes multiple such cells, and a reference to “its peptide” includes one or more peptides and their equivalents known to those skilled in the art, such as polypeptides.

[0039] The publications discussed herein are provided solely for the purpose of disclosing publications prior to the filing date of this application. This specification should not be construed as indicating that the present invention has no prior rights to such publications. Furthermore, the dates of the provided publications may differ from the actual publication dates, and it may be necessary to separately verify the actual publication dates.

[0040] Throughout this disclosure, amino acids will be referred to according to either single-letter or three-letter abbreviations. For the reader's convenience, the single-letter and three-letter abbreviations for amino acids are shown in Table 5 below.

[0041] (Table 5) Abbreviations of amino acids TIFF2026086659000015.tif110128

[0042] Standard methods in molecular biology are described in the scientific literature (see, for example, Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY. These discuss cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). Scientific literature describes protein purification methods including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, fusion protein generation, and protein glycosylation (see, for example, Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0043] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0044] Activate: As used herein, the term “activate” is used to reflect the biological effects on a receptor or receptor complex, both directly and / or through its involvement in a multi-component signaling cascade resulting from the binding of an agonist ligand to a ligand-binding-responsive receptor.

[0045] Activity: As used herein, the term “activity” is used to describe a molecule’s properties in relation to a test system (e.g., an assay), or the biological or chemical properties (e.g., the degree of binding between molecules) or physical properties (e.g., modification of cell membrane potential) of a material or cell. Examples of such biological functions include, but are not limited to, the catalytic activity of a biological agent, its ability to modulate intracellular signaling, gene expression, cell proliferation, and immunological activity such as inflammatory responses. “Activity” is typically expressed as the level of biological activity per unit of a test agent, e.g., [catalytic activity] / [mg protein], [immunological activity] / [mg protein], international units (IU) of activity, [STAT5 phosphorylation] / [mg protein], [proliferation] / [mg protein], plaque-forming units (pfu), etc. As used herein, the term proliferative activity refers to activity that promotes cell proliferation and replication, including dysregulated cell division, such as dysregulated cell division observed in neoplasms, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.

[0046] Administer / Administer: The terms “administer” and “administer” are used herein synonymously to refer to any act of contact with a subject, including, in vitro, in vivo, or ex vivo, the cells, tissues, organs, or biological fluids of the subject, and an agent (e.g., an IL10Rb-binding molecule or engineered cells expressing an IL10Rb-binding molecule, a chemotherapeutic agent, an antibody, or a pharmaceutical formulation comprising one or more of the foregoing). The administration of the agent can be accomplished by any of the various methods recognized in the art, including, but not limited to, local administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal delivery, transmucosal delivery, iontophoresis delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g., respiratory inhalers including dry powder inhalers), intraocular injection, intraperitoneal injection, intrafocal injection, intraovarian injection, intracerebral or intracerebral injection, intraventricular injection (ICVI), etc. The term "administration" includes contact between the agent and cells, tissues, or organs, as well as contact between the agent and fluids in contact with cells, tissues, or organs.

[0047] Affinity: As used herein, the term "affinity" refers to the degree of specific binding between a first molecule (e.g., a ligand) and a second molecule (e.g., a receptor), and the dissociation rate constant (K) of the molecule and its target. off ) and the association rate constant (K) of the molecule and its target. on The equilibrium dissociation constant (K) is the ratio of ). D ) is measured by.

[0048] Agonist: As used herein, the term “agonist” refers to a first agent that specifically binds to a second agent (the “target”) and interacts with the target to cause or promote increased activation of the target. In some cases, an agonist is an activator of a receptor protein that modulates, enhances, increases the cell’s sensitivity to activation by the second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways, or pathways that result in cell cycle arrest or cell death, such as apoptosis. In some embodiments, an agonist is an agent that binds to a receptor and alters its receptor state, thereby resulting in a biological response that mimics the effect of the receptor’s endogenous ligand. The term “agonist” includes partial agonists, full agonists, and superagonists. An agonist may be called a “full agonist” or a partial agonist when such an agonist leads to a substantially complete biological response induced by the receptor under study (i.e., a response related to the innate ligand / receptor binding interaction). A "superagonist" is a type of agonist that can produce a maximal response to a target receptor that exceeds that of an endogenous agonist, and therefore has more than 100% of the activity of the native ligand. Superagonists are typically synthetic molecules that, when evaluated at similar concentrations in comparable assays, exhibit more than 110%, 120%, 130%, 140%, 150%, 160%, or 170% of the response of the native molecule to an evaluable quantitative or qualitative parameter. It should be noted that the biological effects associated with a full agonist may differ in degree and / or type from the biological effects of a partial agonist or superagonist. In contrast to agonists, antagonists can bind specifically to a receptor but do not trigger a signal cascade, typically a signal cascade initiated by the receptor, and may modify the agonist action at that receptor. An inverse agonist is a drug that produces a pharmacological response opposite to that of the agonist.

[0049] Antagonist: As used herein, the terms “antagonist” or “inhibitor” refer to molecules that counteract the action of an agonist. Antagonists block, reduce, inhibit, or neutralize the activity of an agonist, and even in the absence of a specific agonist, antagonists may also block, inhibit, or reduce the constitutive activity of a target, such as a target receptor. Inhibitors are molecules that reduce, block, block, delay, or inactivate, desensitize, or downregulate the activation of a biological pathway, such as a gene, protein, ligand, receptor, immune checkpoint pathway, or a cell, or a biological pathway, such as a gene, protein, ligand, receptor, immune checkpoint pathway, or a cell.

[0050] antibody: As used herein, the term “antibody” collectively refers to (a) glycosylated or non-glycosylated immunoglobulins that specifically bind to a target molecule, and (b) antibody fragments such as single-domain antibodies, including but not limited to those immunoglobulin derivatives. In some embodiments, the immunoglobulin derivative competes with the originating immunoglobulin for binding to the target molecule. The term “antibody” is not limited to immunoglobulins derived from any particular species, but includes antibodies from cartilaginous fish, including but not limited to mice, humans, horses, camelids, and sharks. The term “antibody” includes antibodies that can be isolated from natural sources or animals after immunization with an antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR graft antibodies, veneered antibodies, or deimmunized (e.g., aimed at removing T cell epitopes) antibodies, camelidized (in the case of VHH), or molecules containing an antibody binding domain (e.g., CDR) in a non-immunoglobulin scaffold. The term “antibody” should not be interpreted as being limited to any particular synthetic means, and includes natural antibodies that can be isolated from natural sources, as well as manipulated antibody molecules prepared by “recombinant” means, including antibodies isolated from transgenic animals into which human immunoglobulin genes have been introduced or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs resulting in antibody expression, and antibodies isolated from combinatorial antibody libraries, including phage display libraries. In one embodiment, “antibody” is a mammalian immunoglobulin of the IgG1, IgG2, IgG3, or IgG4 class. In some embodiments, the antibody is a “full-length antibody” containing variable and constant domains that provide binding and effector functions. As used herein, the term “single-domain antibody” (sdAb) refers to an antibody fragment consisting of a monomeric variable antibody domain that specifically binds to an antigen and can compete for binding with the parental antibody from which it originated. The term “single-domain antibody” includes scFv and VHH molecules.As used herein, the term "VHH" typically refers to single-domain antibodies derived from camelid antibodies obtained from immunization of camelid animals (including camels, llamas, and alpacas) (see, for example, Hamers-Casterman, et al. (1993) Nature 363:446-448). VHH are also called heavy-chain antibodies or Nanobodies®. Single-domain antibodies may also be derived from non-mammalian sources, such as VHH obtained from IgNAR antibody immunization of cartilaginous fish, including but not limited to sharks.

[0051] Biological samples: As used herein, the terms “biological sample” or “sample” refer to a sample obtained from (or derived from) a subject. For example, a biological sample includes material selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, and tumor tissue, including immunoglobulin-enriched fractions or cell-type-specific, enriched fractions derived from one or more of these tissues.

[0052] IL10Rb cells: The terms “IL10Rb cells,” “IL10Rb-expressing cells,” “IL10Rb-positive cells,” and “IL10Rb+” cells are used herein synonymously to refer to cells that express and show the IL10Rb antigen on the extracellular surface of the cell membrane. Similarly, the terms “IL10Rb-negative cells” and “IL10Rb- cells” are used herein synonymously to describe cells that do not express and show the IL10Rb antigen on their cell surface.

[0053] CDR: As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean discontinuous antigen-binding sites found within the variable regions of both heavy-chain immunoglobulin polypeptides and light-chain immunoglobulin polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, et al., “Sequences of proteins of immunological interest” in a U.S. Department of Health and Human Services publication (1991) (also referred herein as “Kabat 1991” or “Kabat”); Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (also referred herein as “Chothia”); and MacCallum, et al. (1996) J. Mol. Biol. 262:732-745, and the definition includes overlaps or subsets of amino acid residues when compared to one another. Nevertheless, the application of either definition to refer to the CDR of an antibody or a grafted antibody or its variant is intended to be within the scope of the terms as defined and used herein. The term “Chothia numbering” as used herein is recognized in the art and refers to a system of numbering amino acid residues based on the position of the structural loop region (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For the purposes of this disclosure, unless otherwise specifically identified, the positions of CDR2 and CDR3 in the variable region of the antibody follow Kabat numbering or simply “Kabat”. The position of CDR1 in the variable region of the antibody follows a hybrid of the Kabat and Chothia numbering schemes.

[0054] Chronotype : As used herein, chronotype refers to a collection of binding molecules originating from the same B cell progenitor cell. The term “chronotype” is used to refer to a collection of antigen-binding molecules that belong to the same germline family, have the same CDR3 length, and have 70% or higher homology in their CDR3 sequences.

[0055] Equivalent: As used herein, the term “equivalent” is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements would be considered “equivalent” if the first measurement of an evaluable quantitative parameter and the second measurement of the evaluable parameter do not deviate beyond what a person skilled in the art would recognize as not producing a statistically significant difference in effect between the two results in this context. In some cases, measurements may be considered “equivalent” if one measurement deviates from another by less than 35%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 4%, 3%, 2%, or 1%. In certain embodiments, a measurement is equivalent to a standard if it deviates from the standard by less than 15%, 10%, or 5%.

[0056] Conservative amino acid substitutions: As used herein, the term “conservative amino acid substitution” refers to an amino acid exchange in which a particular amino acid is replaced by another amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, the amino acids in the following groups: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid can be considered conserved amino acids of each other.

[0057] Derived from: As used herein, the term "derived from" is intended to indicate, in the context of amino acid sequences, that a polypeptide or nucleic acid has a sequence based on the sequence of a reference polypeptide or nucleic acid, and is not intended to be limited to the source or method by which the protein or nucleic acid is produced. For example, the term "derived from" includes homologs or variants of a reference amino acid sequence or DNA sequence.

[0058] Effective concentration (EC): As used herein, the term “effective concentration” or its abbreviation “EC” is used synonymously to refer to a concentration of an agent sufficient to alter a particular parameter in a test system. The abbreviation “E” refers to the magnitude of a particular biological effect observed in the test system when the test system is exposed to the test agent. The abbreviation “EC” is used when the magnitude of the response is expressed as a factor of the concentration of the test agent ("C"). In the context of biological systems, the term Emax refers to the maximum magnitude of a particular biological effect observed in response to the saturation concentration of the activating test agent. When the abbreviation EC is shown with a subscript (e.g., EC), 40 , EC 50 (e.g.), the subscript indicates the percentage of the Emax of the biological response observed at this concentration. For example, 30% of the maximum level of such a measurable biological parameter in response to such a test agent is the concentration of the test agent sufficient to induce a measurable biological parameter in the test system, which is 30% of the maximum level of such a measurable biological parameter in response to such a test agent. 30 It is called "EC". 100 The term "EC" is used to indicate the effective concentration of an agent that yields the maximum (100%) response of a measurable parameter in response to such an agent. Similarly, (commonly used in the field of pharmacokinetics) EC 50The term refers to the concentration of an agent sufficient to bring about a maximal half change (about 50%) in a measurable parameter. The term "saturation concentration" refers to the maximum amount of a test agent that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacokinetics, the saturation concentration of a drug is typically used to indicate a sufficient concentration of the drug such that all available receptors are occupied by the drug, and EC 50 is the drug concentration that produces a maximal half effect.

[0059] Concentrated: As used herein, the term "enriched" refers to a sample that has been non-naturally engineered such that (a) the species of interest (e.g., a molecule or a cell) is present at a concentration higher (e.g., at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold) than the concentration of that species in a starting sample, e.g., a biological sample (e.g., a sample in which the molecule naturally exists, or a sample that exists after administration), or (b) the molecule is present at a concentration higher than the environment in which it was made (e.g., a recombinantly modified bacterial or mammalian cell).

[0060] Extracellular domain: As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is outside the plasma membrane of a cell. The cell surface protein may be a transmembrane protein, a cell surface protein, or a membrane-bound protein.

[0061] Identity: As used herein with respect to polypeptide sequences or DNA sequences, the term “identity” refers to subunit sequence identity between two molecules. If the subunit positions of both molecules are occupied by the same monomeric subunits (i.e., the same amino acid residues or nucleotides), then the molecules are identical at that position. Similarity between two amino acid sequences or two nucleotide sequences is a linear function of the number of identical positions. Generally, these sequences are aligned to obtain the highest-order match. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the BLAST 2.0 algorithm described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or fit a positive threshold score "T" when aligned with words of the same length in the database sequence. T is called the neighbor word score threshold (Altschul et. al., previously mentioned). These initial neighbor word hits act as a seed to initiate a search for longer HSPs that contain them. These word hits are then extended bidirectionally along each sequence as long as the cumulative alignment score increases. For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for a pair of matched residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.(a) when the cumulative alignment score decreases by amount X from its maximum attainable value; when the cumulative score becomes 0 or less due to the accumulation of one or more negative score residue alignments; or (b) when the end of either sequence is reached, the extension of word hits in each direction stops. The BLAST algorithm parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) works similarly, but uses a word size of 28 ("W"), an expected value of 10 ("E"), M=1, N=-2, and comparison of both strands by default. For amino acid sequences, the BLASTP program uses a word size of 3 ("W"), an expected value of 10 ("E"), and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS(USA) 89:10915-10919 by default).

[0062] In sufficient quantities to elicit a response: As used herein, the phrase “in an amount sufficient to produce a response” refers to an amount of the test agent sufficient to produce a detectable change in the level of an indicator measured before the application of the test agent to the test system (e.g., baseline level) and the level of the indicator measured after application. In some embodiments, the test system is a cell, tissue, or organism. In some embodiments, the test system is an in vitro test system, such as a fluorescence assay. In some embodiments, the test system is an in vivo system involving the measurement of changes in parameter levels of cells, tissues, or organisms that reflect their biological function before and after the application of the test agent to the cells, tissues, or organisms. In some embodiments, the indicators reflect the biological function or developmental state of cells evaluated in the assay in response to the administration of a certain amount of the test agent. In some embodiments, the test system involves the measurement of changes in indicator levels of cells, tissues, or organisms that reflect their biological state before and after the application of one or more test agents to cells, tissues, or organisms. The term “in an amount sufficient to produce a response” may be sufficient if it is a therapeutically effective dose, but may also be greater or less than a therapeutically effective dose.

[0063] Treatment is required : When used herein, the term “requiring treatment” refers to a determination made by a physician or other caregiver regarding a subject that the subject requires treatment or could potentially benefit from treatment. This determination is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0064] Preventive measures are necessary : As used herein, the term “requiring prevention” means a judgment made by a physician or other caregiver regarding a subject that the subject requires or could potentially benefit from preventive care. This judgment is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0065] Inhibitor: As used herein, the term “inhibitor” refers to a molecule that reduces, blocks, inhibits, delays, or inactivates, desensitizes, or downregulates the activation of a gene, protein, ligand, receptor, or cell, for example. An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates the constitutive activity of a cell or organism.

[0066] Intracellular domain: As used herein, the term “intracellular domain” or its abbreviation “ICD” refers to a portion of a cell surface protein (e.g., a cell surface receptor) located inside the plasma membrane of a cell. An ICD may include the entire cytoplasmic portion of a transmembrane protein or membrane-bound protein, or it may include an intracellular protein.

[0067] Isolated: As used herein, the term “isolated” applies to polypeptides of interest that, if naturally occurring, are in an environment different from the environment in which they could naturally occur. “Isolated” is intended to mean that the polypeptide in a sample is fairly concentrated and / or partially or substantially purified. If the polypeptide is not naturally occurring, “isolated” means that the polypeptide has been separated from the environment in which it was synthesized, for example, from a recombinant cell culture containing cells engineered to express the polypeptide, or from a solution resulting from solid-phase synthesis.

[0068] Kabat numbering: As used herein, the term “Kabat numbering” is recognized in the art and refers to a system for numbering amino acid residues in the heavy and light chain regions of immunoglobulins that are more variable than other amino acid residues (e.g., hypervariable) (Kabat, et al., (1971) Ann. NY Acad. Sci. 190:382-93; Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). As used herein, the term “Chothia numbering” is recognized in the art and refers to a system of numbering amino acid residues based on their location in the structural loop region (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For the purposes of this disclosure, unless otherwise specifically identified, the locations of CDR2 and CDR3 in the variable region of the antibody follow Kabat numbering, or simply “Kabat”. The location of CDR1 in the variable region of the antibody follows a hybrid of the Kabat and Chothia numbering schemes.

[0069] Ligand: As used herein, the term “ligand” refers to a molecule that specifically binds to a receptor and causes a change in the receptor to alter the receptor’s activity or the response of a cell expressing that receptor. In one embodiment, the term “ligand” refers to a molecule or complex that can act as an agonist or antagonist of a receptor. As used herein, the term “ligand” encompasses both natural and synthetic ligands. “Ligands” also encompass small molecules, cytokines, and peptidomimetic antibodies. The ligand-receptor complex is called a “ligand-receptor complex.” A ligand may contain one domain of a polyprotein or fusion protein (e.g., one domain of an antibody / ligand fusion protein).

[0070] Adjust: As used herein, terms such as “modulate” and “adjust” refer to the ability of a test agent to elicit, directly or indirectly, a positive or negative response in a system or biochemical pathway, including biological systems. The term “modulator” includes both agonists (including partial agonists, full agonists, and superagonists) and antagonists.

[0071] Nucleic acid: The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used herein synonymously to refer to polymeric forms or analogues of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. Non-exclusive examples of polynucleotides include linear and cyclic nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, and primers.

[0072] Functionally linked: The term "functionally linked" is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, that are arranged within a construct in such a way that the function of each component molecule is preserved, but functional linking can positively or negatively modulate the activity of individual components of the construct. For example, functionally linking polyethylene glycol (PEG) molecules to a wild-type protein may result in a construct in which the biological activity of that protein is reduced compared to the wild-type molecule. Nevertheless, the two are considered functionally linked. When the term "functionally linked" is applied to the relationship between multiple nucleic acid sequences encoding different functions, when multiple nucleic acid sequences are combined to form a single nucleic acid molecule, for example, when this nucleic acid molecule is introduced into a cell using recombination techniques, it provides a nucleic acid that can transcribe and / or translate a specific nucleic acid sequence within the cell. For example, if a nucleic acid sequence encoding a signal sequence that facilitates polypeptide secretion expresses a preprotein, it can be considered functionally linked to the DNA encoding the polypeptide. If a promoter or enhancer affects the transcription of a sequence, it is considered functionally linked to the coding sequence. Alternatively, a sequence is considered functionally linked to a coding sequence if the ribosome binding site is positioned to facilitate translation. Generally, in the context of nucleic acid molecules, the term "functionally linked" means that the linked nucleic acid sequence is contiguous, and in the case of a secretory leader or linked subdomain of the molecule, it is contiguous and in the reading phase. However, certain genetic factors, such as enhancers, may function away from the sequence in which they exert their effect and do not need to be contiguous with respect to that sequence, but can still be considered functionally linked.

[0073] Parent polypeptide: As used herein, the terms “parent polypeptide” or “parent protein” are used synonymously to specify the source of a second polypeptide (e.g., a derivative, mutaine, or variant) that is modified relative to a first “parent” polypeptide. In some cases, the parent polypeptide is a wild-type or native protein. In some cases, the parent polypeptide may be a further modified, modified form of a native protein. The term “parent polypeptide” may refer to the polypeptide itself or a composition containing the parent polypeptide (e.g., a glycosylated or PEGylated form and / or a fusion protein containing the parent polypeptide).

[0074] Partial agonist: As used herein, the term “partial agonist” refers to a molecule that specifically binds to and activates a particular receptor, but only partially activates it compared to a full agonist. Partial agonists may exhibit both agonist and antagonistic effects. For example, when both a full agonist and a partial agonist are present, the partial agonist competes with the full agonist for receptor binding, and as a result acts as a competitive antagonist by reducing receptor activation compared to contact between the receptor and the full agonist in the absence of the partial agonist. When an insufficient amount of endogenous ligand is present, a partial agonist can be used to activate a receptor to produce a desired submaximal response in a subject. Alternatively, when an excess amount of endogenous ligand is present, a partial agonist can reduce receptor overstimulation. The maximum response (E) produced by a partial agonist maxThis is called the intrinsic activity and is sometimes expressed on a percentage scale when a complete agonist produces a 100% response. Partial agonists may have more than 10% but less than 100% of the activity of a reference polypeptide when evaluated at similar concentrations in a particular assay system, or more than 20% but less than 100%, or more than 30% but less than 100%, or more than 40% but less than 100%, or more than 50% but less than 100%, or more than 60% but less than 100%, or more than 70% but less than 100%, or more than 80% but less than 100%, or more than 90% but less than 100%.

[0075] polypeptide: As used herein, the terms “polypeptide,” “peptide,” and “protein” are synonymous and refer to polymeric forms of amino acids of any length, including amino acids specified by the genetic code and amino acids not specified by the genetic code, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified polypeptide backbone. The term polypeptide includes, but is not limited to, fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins having or not having an N-terminal methionine residue; fusion proteins having amino acid sequences that facilitate the purification of chelated peptides, etc.; fusion proteins having immunologically tagged proteins; and fusion proteins containing peptides having immunologically active polypeptide fragments (e.g., antigenic diphtheria or tetanus toxin or toxoid fragments).

[0076] Prevent : As used herein, terms such as “prevent,” “prevent,” and “prevent” refer to a process of action initiated with respect to an object before the onset of a disease, disorder, condition, or its symptoms, in order to prevent, suppress, inhibit, or reduce (for example, determined by the absence of clinical symptoms) the risk of the object developing a disease, disorder, or condition, whether temporarily or permanently. A process of action to prevent a disease, disorder, or condition in an object typically applies in the context of an object that has a predisposition to developing a disease, disorder, or condition due to genetic, experimental, or environmental factors that cause the development of a particular disease, disorder, or condition. In certain specific cases, the terms “prevent,” “prevent,” and “prevent” are also used to refer to the slowing of the progression of a disease, disorder, or condition from an existing condition to a more harmful one.

[0077] Receptor: As used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds to a ligand, such that binding of the ligand alters at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane-bound protein containing an extracellular domain (ECD) and a membrane-bound domain that helps fix the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide in which an intracellular domain (ICD) and an extracellular domain (ECD) are linked by a transmembrane domain commonly called a transmembrane domain (TM). When a cognitive ligand binds to a receptor, a conformational change occurs in the receptor, resulting in a measurable biological effect. In some cases, if the receptor is a transmembrane polypeptide containing an ECD, TM, and ICD, binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD. In some embodiments, the receptor is a component of a multicomponent complex that facilitates intracellular signaling. For example, a ligand, while not involved in any intracellular signaling on its own, may, upon binding, promote the formation of heteromultimer (including heterodimers, heterotrimers, etc.) or homomultimer (including homodimers, homotrimers, homotetramers, etc.) complexes, resulting in measurable biological effects within the cell, such as activating an intracellular signaling cascade (e.g., the Jak / STAT pathway) on a cell surface receptor. In some embodiments, the receptor is a transmembrane single-chain polypeptide containing ECD, TM, and ICD domains, the ECD, TM, and ICD domains derived from the same or different native receptor variants or their synthetic functional equivalents.

[0078] Recombination: As used herein, the term “recombinant” is used as an adjective to describe the modification of polypeptides, nucleic acids, or cells using recombinant DNA technology. A “recombinant protein” is a protein produced using recombinant DNA technology and is abbreviated with a lowercase “r” before the protein name to indicate the method of production (for example, recombinant human growth hormone is commonly abbreviated as “rhGH”). Similarly, cells are called “recombinant cells” if they have been modified by the incorporation of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.) using recombinant DNA technology (e.g., transfection, transduction, infection). Techniques and protocols for recombinant DNA technology, such as those found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals, are well known in the art.

[0079] response: For example, the term “response” of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in evaluable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy expenditure rate, level or state of differentiation) that correlate with activation, stimulation, or treatment by internal mechanisms such as exogenous agents or genetic programming, or with contact with such internal mechanisms. In certain contexts, terms such as “activation” and “stimulation” refer to cellular activation regulated by internal mechanisms as well as by external or environmental factors, while terms such as “inhibition” and “downregulation” refer to the opposite effect. “Response” may be evaluated in vitro, for example, by using assay systems, surface plasmon resonance, enzyme activity, mass spectrometry, amino acid or protein sequencing techniques. The “response” may be quantitatively assessed in vivo by evaluating objective physiological parameters, such as body temperature, body weight, tumor volume, blood pressure, X-ray or other imaging techniques, or qualitatively assessed by reported subjective changes in mood, such as happiness, depression, excitement, or pain. In some embodiments, the proliferation level of CD3-activated primary human T cells may be assessed in a bioluminescence assay that generates a luminescence signal proportional to the amount of ATP present, directly proportional to the number of viable cells present in culture, as described in Crouch, et al. (1993) J. Immunol. Methods 160: 81-8, or it may be assessed using a commercially available assay, such as the CellTiter-Glo® 2.0 Cell Viability Assay or CellTiter-Glo® 3D Cell Viability Kit, commercially available from Promega Corporation, Madison WI 53711 as catalog numbers G9241 and G9681, generally in accordance with the instructions provided by the manufacturer.In some embodiments, the T cell activation level in response to the administration of the test agent may be determined by flow cytometry, as described when it is determined by STAT (e.g., STAT1, STAT3, STAT5) phosphorylation levels according to methods well known in the art.

[0080] Significantly reduced binding: As used herein, the term “showing significantly reduced binding” applies to a variant of a first molecule (e.g., ligand or antibody) that shows a significant reduction in affinity to a second molecule (e.g., receptor or antigen) compared to the parent form of the first molecule. With respect to an antibody variant, the antibody variant “shows significantly reduced binding” if it binds to the native receptor with an affinity of less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5%, of the parent antibody from which the variant originated. Similarly, with respect to a variant ligand, the variant ligand “shows significantly reduced binding” if its affinity binds to the receptor with an affinity of less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5%, of the parent ligand from which the variant ligand originated. Similarly, with respect to variant receptors, if the affinity of the variant receptor is less than 20%, or about 10%, or about 8%, or about 6%, or about 4%, or about 2%, or about 1%, or about 0.5%, of the affinity of the parent receptor from which the variant receptor originated, then the variant ligand "shows significantly reduced binding."

[0081] Small molecules: The term "low molecular weight" refers to compounds with molecular weights of less than approximately 10 kDa, less than approximately 2 kDa, or less than approximately 1 kDa (typically pharmaceutically active compounds). Low molecular weight includes, but is not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic molecules, molecules containing radioactive atoms, and synthetic molecules. The term "low molecular weight" is well understood by those skilled in the pharmaceutical field and is typically used to distinguish organic compounds from biologics.

[0082] Binds specifically: As used herein, the term “specifically binds” refers to the degree of affinity that a first molecule exhibits for a second molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen), a first molecule of a binding pair is said to specifically bind to its second molecule when it does not bind in significant amounts to other components present in the sample. A first molecule of a binding pair is said to specifically bind to its second molecule when its affinity for the second molecule is at least twice, at least five times, at least ten times, at least twenty times, or at least 100 times, the affinity of the first molecule for other components present in the sample. In certain embodiments, when the first molecule of a binding pair is an antibody, the equilibrium dissociation constant (K) between the antibody and the antigen is used. D For example, when determined by scatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239), it is approximately 10 -6 Less than M, or about 10 -8 Less than M, or about 10 -10 Less than M, or about 10 -11 Less than M, approximately 10 -12 If the M value is less than M, the antibody specifically binds to the antigen (or the antigenic determinant (epitope) of a protein, antigen, ligand, or receptor). In one embodiment, when the ligand is IL10Rb-bound sdAb and the receptor contains IL10Rb, the equilibrium dissociation constant of IL10Rb-bound sdAb / IL10Rb ECD is approximately 10 5 Over M, or about 10 6Over M, or about 10 7 Over M, or about 10 8 Over M, or about 10 9 Over M, or about 10 10 M or more, or about 10 11 If the M value is greater than M, IL10Rb-bound sdAb will bind specifically. Specific binding can be evaluated using techniques known in the art, including but not limited to competitive ELISA assays, radioactive ligand binding assays (e.g., saturated binding, scatchard plots, non-linear curve fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)); and surface plasmon resonance assays (see, for example, Drescher et al., (2009) Methods Mol Biol 493:323-343 and commercially available measuring instruments, e.g., Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA 01752)). In some embodiments, the disclosure provides molecules that specifically bind to hIL10Rb isoforms (e.g., IL10Rb-binding sdAb).

[0083] In this specification, the binding affinity of an IL10Rb-binding molecule to IL10Rb may be determined and / or quantified by surface plasmon resonance ("SPR"). When evaluating the binding affinity of an IL10Rb-binding molecule to IL10Rb, one member of the binding pair may be immobilized, and the other element of the binding pair may be provided in the mobile phase. In some embodiments, a sensor chip on which the protein of interest is immobilized is conjugated with a substance that facilitates the binding of the protein of interest, such as a nitrilotriacetate (NTA) derivatized surface plasmon resonance sensor chip (e.g., Sensor Chip NTA, available from Cytiva Global Life Science Solutions USA LLC, Marlborough MA as catalog number BR100407), an anti-His tag antibody (e.g., the anti-histidine CM5 chip, commercially available from Cytiva, Marlborough MA), protein A, or biotin. As a result, it is often necessary to modify the protein to bind to the substance conjugated on the chip surface in order to evaluate the binding. For example, one member of the binding pair to be evaluated was conjugated by NTA by incorporating a chelated peptide containing a polyhistidine sequence (e.g., 6xHis (SEQ ID NO: 170) or 8xHis (SEQ ID NO: 171)) for retention on the chip. In some embodiments, the IL10Rb binding molecule may be immobilized on the chip, and IL10Rb (or its ECD fragment) may be provided in the mobile phase. Alternatively, IL10Rb (or its ECD fragment) may be immobilized on the chip, and the IL10Rb binding molecule may be provided in the mobile phase. In either case, it should be noted that modifying some proteins for immobilization on a coated SPR chip may interfere with the binding properties of one or both components of the binding pair to be evaluated by SPR. In such cases, it may be necessary to switch the mobile and binding elements of the binding pair, or to use a chip with a binder that facilitates non-interfering conjugation of the protein to be evaluated.Alternatively, when evaluating the binding affinity of an IL10Rb-binding molecule to IL10Rb using SPR, the IL10Rb-binding molecule may be derivatized by C-terminal addition of a polyHis sequence (e.g., 6xHis (SEQ ID NO: 170) or 8xHis (SEQ ID NO: 171)) and immobilized on an NTA derivatized sensor chip, and the hIL10Rb receptor subunit whose binding affinity is being evaluated is provided in the mobile phase. Means for incorporating a polyHis sequence into the C-terminus of an IL10Rb-binding molecule produced by recombinant DNA technology are well known to those skilled in the art in the relevant fields of biotechnology. In some embodiments, the binding affinity of an IL10Rb-binding molecule to IL10Rb is evaluated using SPR in general accordance with the disclosure of examples.

[0084] subject: The terms “recipient,” “individual,” “subject,” and “patient” are used synonymously herein and refer to any mammalian subject, in particular human, for which diagnosis, treatment, or therapy is desired. For treatment purposes, “mammal” refers to any animal classified as a mammal, including humans, domesticated and livestock, and zoo, sport, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, and pigs. In some embodiments, mammal is human.

[0085] Essentially pure: As used herein, the term “substantially pure” means that one component of the composition constitutes more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% of the total content of the composition. “Substantially pure” protein constitutes more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% of the total content of the composition.

[0086] ~ is suffering from : As used herein, the term "suffering from" refers to a determination made by a physician regarding a subject that the subject requires or would benefit from treatment, based on available objective or subjective information acceptable in the field of identifying a disease, disorder, or condition, including but not limited to X-ray, CT scan, conventional laboratory diagnostic tests (e.g., blood count), genomic data, protein expression data, and immunohistochemistry. The term "suffering from" is typically used in conjunction with a specific disease condition; for example, "suffering from a neoplasm" refers to a subject diagnosed with the presence of a neoplasm.

[0087] T cells: As used herein, the term “T cell” (or “T-cell”) is used in its conventional sense to refer to lymphocytes that differentiate in the thymus, possess specific cell surface antigen receptors, and control the initiation or suppression of cellular and humoral immunity, and that lyse antigen-bearing cells. In some embodiments, T cells include naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, for example, T H 1. T H 2, T H 9, T H 11, T H 22, T FH ; regulatory T cells, e.g. T R 1. Tregs, inducible Tregs; memory T cells, such as central memory T cells, effector memory T cells, NKT cells, tumor-infiltrating lymphocytes (TILs), and engineered variants of such T cells, including but not limited to CAR-T cells, recombinant modified TILs, and TCR-manipulated cells. In some embodiments, T cells are T cells expressing an IL10Rb isoform, referred to synonymously with IL10Rb cells, IL10Rb+ cells, IL10RbT cells, or IL10Rb+T cells.

[0088] Terminus / Terminal: In the context of polypeptide structures, the terms “N-terminus” (or “amino-terminus”) and “C-terminus” (or “carboxyl-terminus”) as used herein refer to the amino-terminus and carboxyl-terminus of the polypeptide, respectively. In contrast, the terms “N-terminus” and “C-terminus” refer to the relative positions in the polypeptide amino acid sequence to the N-terminus and C-terminus, respectively, and may include residues located at the N-terminus and C-terminus, respectively. “Immediately N-terminal” refers to the position of the first amino acid residue relative to the second amino acid residue in a contiguous polypeptide sequence, where the first amino acid is close to the N-terminus of the polypeptide. “Immediately C-terminal” refers to the position of the first amino acid residue relative to the second amino acid residue in a contiguous polypeptide sequence, where the first amino acid is close to the C-terminus of the polypeptide.

[0089] Therapeutic effective dose : As used herein, the term “therapeutic dose” refers to the amount of an agent, administered to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, that provides a positive effect on any quantitative or qualitative symptom, aspect, or feature of a disease, disorder, or condition. The therapeutic dose can be determined by measuring the relevant physiological effect, which may be adjusted in connection with the drug regimen and in response to a diagnostic analysis of the subject’s condition. Parameters for evaluation to determine the therapeutic dose of an agent are determined by a physician using diagnostic criteria acceptable in the art, including but not limited to signs such as age, weight, sex, overall health status, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, and X-ray. Alternatively or additionally, other parameters commonly evaluated in clinical settings may be monitored to determine whether a therapeutically effective dose of the agent has been administered to the subject, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptoms, aspects, or characteristics of the disease, disorder, or condition, biomarkers (e.g., inflammatory cytokines, IFN-γ, and granzymes), reduction in serum tumor markers, improvement in Response Evaluation Criteria In Solid Tumors (RECIST), improvement in Immune-Related Response Criteria (irRC), increase in duration of survival, extension of duration of progression-free survival, extension of time to progression, increase in time to treatment failure, extension of duration of event-free survival, extension of time to the next intervention, improvement in objective response rate, improvement in duration of response, reduction in tumor burden, complete response, partial response, stable disease, and similar assessments of improvement in the subject's condition in response to the administration of the agent.In one embodiment, a therapeutically effective dose is the amount of an agent, when used alone or in combination with another agent, that provides a positive effect on any quantitative or qualitative symptom, aspect, or feature of a disease, disorder, or condition, and that does not result in any irreversible serious adverse event during the course of administration of the agent to a mammalian subject.

[0090] Transmembrane domain: The term “transmembrane domain” or “TM” refers to the polypeptide domain of a transmembrane polypeptide (e.g., a transmembrane receptor) that is embedded in the cell membrane when the transmembrane polypeptide is bound to the cell membrane and is peptidyl-bound to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide. The transmembrane domain may be homogeneous (naturally related) or heterogeneous (naturally unrelated) to either or both of the extracellular and / or intracellular domains. In some embodiments, if the receptor is a chimeric receptor containing an intracellular domain derived from a first parent receptor and the second extracellular domain derived from a second different parent receptor, the transmembrane domain of the chimeric receptor is typically the transmembrane domain associated with either the ICD or ECD of the parent receptor from which the chimeric receptor originated.

[0091] To treat: The terms “to treat,” “to treat,” and “treatment” refer to a course of action initiated with respect to a subject in response to a diagnosis that the subject is suffering from a disease, disorder, or condition or symptoms thereof (e.g., contact between the subject and a pharmaceutical composition containing IL10Rb-binding sdAb alone or in combination with an adjuvant), and the course of action is initiated to (a) the underlying cause of such disease, disorder, or condition that is afflicting the subject; and / or (b) to temporarily or permanently eliminate, reduce, suppress, alleviate, or remit at least one of the symptoms associated with such disease, disorder, or condition. In some embodiments, treatment includes a course of action taken with respect to a subject suffering from a disease, and the course of action inhibits the disease of the subject (e.g., the development of the disease, disorder, or condition is suppressed, or one or more symptoms associated therewith are remitted).

[0092] Treg cells or regulatory T cells: The terms "regulatory T cells," "Treg cells," or "Treg" refer to effector T cells (T eff CD4, which can suppress other T cell responses, including but not limited to these. + In this specification, the terms "Treg" and "CD4+" are used synonymously to refer to a type of T cell. Treg cells are typically characterized by the expression of CD4 (CD4+), the CD25 subunit of the IL2 receptor (CD25+), and the transcription factor forkhead box P3 (FOXP3+) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)). In some cases, the term "conventional CD4+ T cell" is used synonymously with "non-Treg CD4+". + T cells CD4 + This is used to distinguish it from Treg.

[0093] Variant: The terms “variant,” “protein variant,” “variant protein,” or “variant polypeptide” are used herein synonymously to refer to a polypeptide that differs from a parent polypeptide by at least one amino acid modification, substitution, or deletion. The parent polypeptide may be a natural or wild-type (WT) polypeptide, or a modified version of a WT polypeptide. The term “variant polypeptide” may refer to the polypeptide itself, a composition containing the polypeptide, or a nucleic acid sequence encoding it. In some embodiments, a variant polypeptide may involve about 1 to about 10, or about 1 to about 8, or about 1 to about 7, or about 1 to about 5, or about 1 to about 4, or about 1 to about 3, or 1 to about 2 amino acid modifications, substitutions, or deletions compared to the parent polypeptide, or a modification, substitution, or deletion of one amino acid. The variant may be at least about 99% identical, or at least about 98% identical, or at least about 97% identical, or at least about 95% identical, or at least about 90% identical to the parent polypeptide from which the variant originated.

[0094] VHH : When used herein, "V H The term "H" refers to a type of sdAb having a single monomeric weight-chain variable antibody domain. Such antibodies may be found in camelid mammals (e.g., camels, llamas) that naturally lack a light chain, or may be produced in such animals. H H is obtained from immunization of camelids (including camels, llamas, and alpacas) (see, for example, Hamers-Casterman, et al. (1993) Nature 363: 446-448), or V H Antibodies with a given specificity may also originate from non-mammalian sources, such as V H H can be obtained from immunization of cartilaginous fish, including but not limited to sharks. In certain embodiments, the dual specificity V described herein H H2 V in the binding molecule H H is V H The equilibrium dissociation constant (K) between H and the receptor. D ) is determined, for example, by scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107: 220-239), approximately 10 -6 Lower than M, or about 10 -8 Lower than M, or about 10 -10 Lower than M, or about 10 -11 Lower than M, or about 10 -10 Lower than M, approximately 10 -12 When M is lower than M, it binds to the receptor (e.g., the first or second receptor of a native or unnative receptor pair). Standardized protocols for the production of single-domain antibodies from camelids are well known in the scientific literature. For example, Vincke, et al (2012) Chapter 8 in Methods in Molecular Biology See Walker, J. editor (Humana Press, Totowa NJ). Specific binding may be evaluated using techniques known in the art, including but not limited to competitive ELISA, BIACORE® assay and / or KINEXA® assay. In some embodiments, V described herein H H can be humanized to include the human framework domain. Humanized V H Examples of human germlines that can be used to produce H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).

[0095] Wild type: In this specification, "wild-type," "WT," or "native" refers to amino acid or nucleotide sequences found in nature, including allele variations. Wild-type proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., have amino acid or nucleotide sequences that have not been modified by humans.

[0096] IL10Rb The IL10Rb binding molecule of this disclosure specifically binds to the extracellular domain of IL10Rb.

[0097] Human IL10Rb: In one embodiment, IL10Rb is human IL10Rb (hIL10Rb). hIL10Rb is expressed as a 325-amino acid preprotein, the first 19 amino acids containing a signal sequence that is post-translationally cleaved in the mature 306-amino acid protein. Amino acids 20-220 (amino acids 1-201 in the mature protein) correspond to the extracellular domain, amino acids 221-242 (amino acids 202-223 in the mature protein) correspond to the 22-amino acid transmembrane domain, and amino acids 243-325 (amino acids 224-306 in the mature protein) correspond to the intracellular domain. hIL10Rb is referenced as entry Q08334 in the UniProtKB database. The canonical full-length hIL10Rb precursor has the following amino acid sequence: This is a polypeptide containing TIFF2026086659000016.tif38134.

[0098] To generate sdAb against human IL10Rb, the extracellular domain of the hIL10Rb protein is used as an immunogen. The extracellular domain of mature (lacking a signal sequence) hIL10Rb has the following amino acid sequence: It has TIFF2026086659000017.tif24132.

[0099] For the purposes of this disclosure, the numbering of amino acid residues of the human IL10Rb polypeptide described herein is done according to the numbering of this canonical sequence (UniProt ID: Q08334). Amino acids 1-19 of SEQ ID NO: 166 are identified as the signal peptide of IL10Rb, amino acids 20-220 of SEQ ID NO: 166 are identified as the extracellular domain, amino acids 221-242 of SEQ ID NO: 1 are identified as the transmembrane domain, and amino acids 243-325 of SEQ ID NO: 166 are identified as the intracellular domain.

[0100] Mouse IL10Rb In one embodiment, IL10Rb is mouse IL10Rb. Mouse IL10Rb (mIL10Rb) is expressed as a 349-amino acid preprotein containing a 19-amino acid N-terminal signal sequence. Amino acids 20-220 (amino acids 1-201 in the mature protein) correspond to the extracellular domain, amino acids 221-241 (amino acids 202-222 in the mature protein) correspond to a 21-amino acid transmembrane domain, and amino acids 242-349 (amino acids 223-330 in the mature protein) correspond to the intracellular domain. mIL10Rb is referenced as entry Q61190 in the UniProtKB database.

[0101] The canonical full-length mRNA10Rb precursor protein, including the signal sequence, has the following amino acid sequence: This is the polypeptide of TIFF2026086659000018.tif38135.

[0102] To generate sdAb against mIL10Rb, the extracellular domain of the mIL10Rb protein is used as an immunogen. The extracellular domain of mature (lacking signal sequence) hIL10Rb has the amino acid sequence (amino acids 27-240): It has TIFF2026086659000019.tif24134.

[0103] For the purposes of this disclosure, the numbering of amino acid residues of the mouse IL10Rb polypeptide described herein is performed according to the numbering of this canonical sequence (UniProt ID: Q61190). Amino acids 1-19 of SEQ ID NO: 168 are identified as the IL10Rb signal peptide, amino acids 20-220 of SEQ ID NO: 168 are identified as the extracellular domain, amino acids 221-241 of SEQ ID NO: 168 are identified as the transmembrane domain, and amino acids 242-349 of SEQ ID NO: 168 are identified as the intracellular domain.

[0104] IL10Rb binding molecule and single-domain antibody In some embodiments, the IL10Rb-binding molecule of this disclosure is a single-domain antibody (sdAb). This disclosure relates to an IL10Rb-binding molecule comprising a single-domain antibody (sdAb) that specifically binds to the extracellular domain of human IL10Rb isoforms (hIL10Rb) found on all IL10Rb-expressing cells.

[0105] A single-domain antibody (sdAb) is an antibody containing a single monomeric variable antibody domain. Like full-length antibodies, sdAbs can specifically bind to antigenic determinants. hIL10Rb-conjugated VHH single-domain antibodies can be engineered from heavy-chain antibodies isolated from camelid mammals (e.g., camels, llamas, dromedaries, alpacas, and guanacos) immunized with the extracellular domain of hIL10Rb or its immunologically active fragment. For descriptions of sdAbs and VHH, see, for example, De Greve et al., (2019) Curr Opin Biotechnol. 61:96-101; Ciccarese, et al., (2019) Front Genet. 10:997; Chanier and Chames (2019). Antibodies ( Basel)8(1); and De Vlieger, et al. (2018) Antibodies (This can be found in Basel)8(1). Alternatively, hIL10Rb single-domain antibodies may be engineered from heavy-chain antibodies isolated from IgNAR heavy-chain antibodies isolated from cartilaginous fish immunized with the extracellular domain of hIL10Rb or its immunoactive fragment. hIL10Rb-conjugated sdAbs may also be obtained by separating the dimer-variable domain from immunoglobulin G (IgG) isotypes from other mammalian species, including humans, rats, and rabbits, immunized with the extracellular domain of hIL10Rb or its immunoactive fragment. While most studies on sdAbs are currently based on heavy-chain variable domains, sdAbs derived from light chains have also been shown to specifically bind to target proteins containing antigen-immunizing sequences. Moller et al., J Biol Chem. 285(49):38348-38361, 2010.

[0106] In some embodiments, the sdAb is a VHH. A VHH is a type of sdAb that has a single monomeric weight-chain variable antibody domain. Similar to traditional antibodies, VHHs can specifically bind to specific antigens. Exemplary VHHs have a molecular weight of about 12–15 kDa, which is much smaller than traditional mammalian antibodies (150–160 kDa) composed of two heavy chains and two light chains. VHHs can be found in camelid mammals that naturally lack light chains (e.g., camels, llamas, dromedaries, alpacas, and guanacos) or can be produced from these animals.

[0107] experiment The single-domain antibodies of this disclosure were obtained from camels by immunization of the extracellular domain of the human IL10Ra receptor (IL10Rb). The IL10Rb VHH molecules of this disclosure were generated substantially according to the teachings of the examples. Briefly, camels were sequentially immunized over several weeks with ECD of human IL10Rb and mouse IL10Rb using a subcutaneous adjuvant-added composition containing a recombinant fusion protein comprising the extracellular domain of IL10Rb, the human IgG1 hinge domain, and the human IgG1 heavy chain Fc. After immunization, RNA extracted from appropriately sized VHH-hinge-CH2-CH3 blood samples was transcribed to generate DNA sequences, digested, and a fragment of approximately 400 bp containing the nucleic acid sequence encoding the VHH domain was identified and isolated. The isolated sequences were digested with restriction endonucleases to induce insertion of the his-tag encoding sequence into an in-frame phagemid vector, and transformation into Escherichia coli (E. coli) was performed to generate a phage library. Multiple rounds of biopanning of the phage library were performed to identify VHHs that bind to the ECD of IL10Rb (human or mouse, as appropriate). Individual phage clones were isolated for periplasmic extract ELISA (PE-ELISA) in a 96-well plate format, and selective binding was confirmed by colorimetric determination. IL10Rb-binding molecules demonstrating specific binding to the IL10Rb antigen were isolated, sequenced, and analyzed to identify VHH sequences, CDRs, and unique VHH chronotypes. As used herein, the term “chronotype” refers to a collection of binding molecules originating from the same B cell progenitor cell, particularly a collection of antigen-binding molecules belonging to the same germline family, having the same CDR3 length, and exhibiting 70% or higher homology in the CDR3 sequence. Table 1 provides VHH molecules demonstrating specific binding to IL10Rb ECD antigen (anti-human IL10Rb VHH) and CDRs isolated from such VHH. Table 3 provides VHH molecules demonstrating specific binding to mIL10Rb ECD antigen (anti-mouse IL10Rb VHH) and CDRs isolated from such VHH.The nucleic acid sequences encoding VHH in Tables 1 and 3 are provided in Tables 2 and 4, respectively.

[0108] To better characterize the binding properties of the VHH molecules generated accordingly and to evaluate their binding affinity, representative examples of each human VHH chronotype were subjected to surface plasmon resonance (SPR) analysis substantially in accordance with the teachings of Example 5 herein. The results of these SPR studies are summarized in Table 6 below.

[0109] (Table 6) Anti-hIL10Rb mono-Fc VHH that binds to hIL10Rb-his (Antigen: Sino Biological, Catalog #10945) TIFF2026086659000020.tif55161* Incorrect Fit

[0110] As shown by the data presented in Table 6, the IL10Rb-binding molecules generated according to the teachings of this disclosure exhibited specific binding to the extracellular domain of IL10Rb and provided a range of affinity.

[0111] In some cases, due to sequence or structural similarities between the extracellular domains of IL10Rb receptors from various mammalian species, immunization with an antigen derived from IL10Rb of a first mammalian species (e.g., IL10Rb-ECD) may produce antibodies that specifically bind to IL10Rb receptors of one or more additional mammalian species. Such antibodies are referred to as "cross-reactive." For example, immunization of camelids with a human-derived antigen (e.g., IL10Rb-ECD) may produce antibodies that cross-reactive to mouse and human receptors. The evaluation of the cross-reactivity of antibodies to receptors derived from other mammalian species can be readily determined by those skilled in the art using methods for evaluating binding affinity and / or specific binding, such as flow cytometry or SPR, as described elsewhere in this specification. Consequently, the use of the terms "human IL10Rb VHH" or "IL10Rb VHH" simply indicates that the species of IL10Rb antigen used for immunization of the camelid animals from which the VHH originated was human IL10Rb (e.g., IL10Rb, ECD, SEQ ID NO: 167), but should not be understood as a limitation regarding the specific binding affinity of VHH to IL10Rb molecules of other mammalian species. Similarly, the use of the terms "mouse IL10Rb VHH" or "mIL10Rb VHH" simply indicates that the species of IL10Rb antigen used for immunization of the camelid animals from which the VHH originated was mouse IL10Rb (e.g., mIL10Rb, ECD, SEQ ID NO: 169), but should not be understood as a limitation regarding the specific binding affinity of VHH to IL10Rb molecules of other mammalian species.

[0112] Modified form of single-domain antibody CDR graft sdAb In some embodiments, the IL10Rb-conjugated sdAb of this disclosure is a CDR-grafted IL10Rb-conjugated sdAb. CDRs obtained from antibodies, heavy chain antibodies, and sdAbs derived therefrom may be grafted onto an alternative framework to generate a CDR-grafted sdAb, as described in Saerens, et al. (2005) J. Mol Biol 352:597-607. In some embodiments, this disclosure provides an IL10Rb-conjugated molecule comprising a CDR-grafted IL10Rb-conjugated sdAb, wherein the CDR-grafted IL10Rb-conjugated sdAb comprises a set of CDR1, 2, and 3 as shown in the row in Table 1 above. In some embodiments, the Disclosure provides an IL10Rb-binding molecule comprising a CDR-grafted IL10Rb-binding sdAb, wherein the CDR-grafted IL10Rb-binding sdAb comprises a set of CDR1, 2, and 3 as shown in the row in Table 3 above.

[0113] Chimeras and humanized sdAbs Any framework region may be used with the CDR described herein. In some embodiments, the IL10Rb-conjugated sdAb is a chimeric sdAb in which the CDR is derived from one species (e.g., camel) and the framework and / or constant region are derived from another species (e.g., human or mouse). In specific embodiments, the framework region is a human or humanized sequence. Thus, a humanized IL10Rb-conjugated sdAb derived from hIL10Rb-conjugated VHH is considered to be within the scope of this disclosure. Techniques for humanizing camelid single-domain antibodies are well known in the art. See, for example, Vincke, et al. (2009) General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold J. Biol. Chem. 284(5) 3273-3284.

[0114] In some embodiments, V described herein H H can be humanized to include the human framework domain. Humanized V H Examples of human germlines that can be used to produce H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).

[0115] Elimination of N-linked glycosylation sites In some embodiments, the amino acid sequence (particularly the CDR sequence) of hIL10Rb-bound sdAb may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser(NXS) or Asn-X-Thr(NXT) (where X is any amino acid other than proline). In such cases, it is desirable to eliminate such N-linked glycosylation motifs by modifying the sequence of the N-linked glycosylation motif to prevent glycosylation. In some embodiments, the elimination of the Asn-X-Ser(NXS)N-linked glycosylation motif may be achieved by incorporating conservative amino acid substitutions of the Asn(N) residue and / or Ser(S) residue of the Asn-X-Ser(NXS)N-linked glycosylation motif. In some embodiments, the exclusion of the Asn-X-Thr(NXT)N-linked glycosylation motif may be achieved by incorporating conserved amino acid substitutions of the Asn(N) and / or Thr(T) residues of the Asn-X-Thr(NXT)N-linked glycosylation motif. In some embodiments, exclusion of the glycosylation site is not required when the IL10Rb-binding molecule is expressed in prokaryotic host cells. Since prokaryotic cells do not provide a mechanism for glycosylation of recombinant proteins, sequence modifications to exclude the N-linked glycosylation site may be unnecessary when using a prokaryotic expression system to produce recombinant IL10Rb-binding sdAb.

[0116] IL10Rb binding molecule containing further agents In some embodiments, the IL10Rb-binding molecules of the present disclosure include an IL10Rb single-domain antibody (sdAb) functionally conjugated to one or more further biologically active agents, including but not limited to combinations of therapeutic agents, chemically active agents, optically active agents, or radioactive materials. A conjugation of at least one such biologically active agent, chemically active agent, optically active agent, or radioactive material confers further biological or chemical properties to the IL10Rb-binding sdAb, and this combination results in an IL10Rb-binding molecule with further utility or different utility.

[0117] For example, further agents may be molecules selected from one or more of the following: immunomodulators (e.g., immunogens); molecules that improve water solubility (e.g., water-soluble polymers and hydrophilic molecules, e.g., sugars); carrier molecules that extend the in vivo half-life (e.g., PEGylation, Fc fusion, or acylation); molecules for use in detection assays (e.g., epitope tags), for ease of purification (e.g., chelated peptides, e.g., polyHis tags), for antibody production; targeting domains that selectively target IL10Rb-binding molecules to specific cell types; therapeutic agents (e.g., therapeutic agents including small molecule or polypeptide agents); and agents that make the molecule more visible to optical or electromagnetic sensors (e.g., radionucleotides or fluorescent substances). In some embodiments, the linker may be a cleavable or incleavable linker. As intended herein, using a cleavable linker in the IL10Rb-binding molecule facilitates the release of the therapeutic agent into the intracellular cytoplasm during the internal translocation of the IL10Rb-binding molecule. Using an incleavable linker would allow for release during digestion of the IL10Rb-binding molecule. Alternatively, a non-cleavable linker could be used with agents that do not require release from the antibody (e.g., imaging agents).

[0118] In some embodiments, the IL10Rb-binding molecule includes an IL10Rb-bonded sdAb that is stably bound to a further agent, linked via a linker. The linker is a covalent bond between the two elements of the IL10Rb-binding molecule (e.g., hIL10Rb-bonded VHH and PEG polymer). The linker may be a covalent bond, a chemical linker, or a peptide linker. A suitable linker generally includes a “mobile linker” of sufficient length to move to some extent between the IL10Rb-bonded sdAb and the linked agent. Examples of chemical linkers include arylacetylenes, ethylene glycol oligomers containing 2-10 monomer units, diamines, dibasic acids, amino acids, or combinations thereof. In some embodiments, the linker is a peptide linker. A suitable peptide linker can be easily selected and may be a linker of any suitable length, for example, a peptide linker with one amino acid (e.g., Gly), two, three, four, five, six, seven, eight, nine, ten, ten-two, twenty-three, thirty-five, or more than fifty amino acids. Suitable peptide linkers are known in the art and include, for example, peptide linkers containing mobile amino acid residues such as glycine and serine. An example of a mobile linker is a glycine polymer (G). n These include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other mobile linkers. Glycine and glycine-serine polymers are relatively structurally indeterminate and therefore can serve as neutral tethers between components. Further examples of mobile linkers include glycine polymer (G) nThese include glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers. Glycine and glycine-serine polymers are relatively structurally indeterminate and therefore may serve as neutral tethers between components. To provide a mobile linker that can be used to conjugate heterogeneous amino acid sequences to the IL10Rb-bound sdAb disclosed herein, polymers of such linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) may be linked together. In some embodiments, the linker has the formula (GGGS)n (SEQ ID NO: 172), (GGGSG)n (SEQ ID NO: 173), or (GGSG)n (SEQ ID NO: 174), where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0119] Immunomodulators In some embodiments, the IL10Rb-binding molecule of the Disclosure is functionally conjugated to an immunomodulator (immunoconjugate). Immunomodulators that can be conjugated to the hIL10Rb-binding sdAb of the Disclosure include, but are not limited to, inactivated viral particles, inactivated bacterial toxins such as toxoids from diphtheria, tetanus, cholera, or leucotoxin molecules, inactivated bacteria, and dendritic cells. Such immunoconjugates are useful in promoting an immune response to IL10Rb or cells expressing IL10Rb.

[0120] Flag tag In one embodiment, the disclosure provides an IL10Rb-binding molecule functionally linked to an antigenic tag such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody as described herein (see, e.g., Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the IL10Rb-binding sdAb polypeptide further comprises a C-terminal c-myc epitope tag.

[0121] Chelated peptides In one embodiment, the present disclosure provides an IL10Rb-binding molecule functionally linked to one or more transition metal chelate polypeptide sequences. As described in U.S. Patent No. 4,569,794 by Smith et al., issued February 11, 1986, the incorporation of such a transition metal chelate domain facilitates purification by immobilized metal affinity chromatography (IMAC). Examples of transition metal chelate polypeptides useful in the implementation of the IL10Rb-binding molecule are described in Smith et al., as mentioned above, and in U.S. Patent No. 5,320,663 by Dobeli et al., issued May 10, 1995. The entirety of these disclosures is incorporated herein by reference. Specific transition metal chelate polypeptides useful in the implementation of the IL10Rb-binding molecule are polypeptides containing 3 to 6 consecutive histidine residues (SEQ ID NO: 175), for example, 6-histidine (His)6 peptide (SEQ ID NO: 170), often referred to in the art as "His tags." In general, following the teachings of Anderson et al. (U.S. Patent No. 5,439,829, issued August 8, 1995) and Hale, JE (1996) Analytical Biochemistry 231(1):46-49, the conjugation of hIL10Rb-binding molecules to such chelated peptides facilitates the targeted delivery of transition metal ions to IL10Rb-expressing cells as kinetically inert or kinetically unstable complexes, in addition to providing a purification "handle" for recombinant proteins or facilitating immobilization on SPR sensor chips. The transition metal ions are reporter molecules, such as fluorescent compounds or radioactive materials including radioimaging agents or radiotherapeutic agents.

[0122] Carrier molecule In some embodiments, the IL10Rb-bound sdAb of this disclosure is functionally linked to one or more carrier molecules. The carrier molecules are typically large, slowly metabolized polymers that provide in vivo stabilization and / or a long duration of action, distinguishing such molecules from conventional carrier molecules used in the preparation of pharmaceutical formulations as described below. Examples of in vivo carriers that can be incorporated into the IL10Rb-bound molecule include, but are not limited to, proteins (including, but not limited to, human serum albumin); fatty acids (acylated); polysaccharides (including, but not limited to, (N-linked and O-linked) sugars, Sepharose, agarose, cellulose, or cellulose); polypeptide amino acid copolymers; acylated or polysialylated polyethylene glycol (PEG) polymers.

[0123] Water-soluble polymers In some embodiments, the IL10Rb-bound sdAb is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the implementation of this IL10Rb-bound molecule include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohol, polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphoren, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0124] Polyethylene glycol In one embodiment, the carrier molecule is a polyethylene glycol ("PEG") polymer. Conjugation (PEGylation) of PEG polymers to proteins is a well-established method for extending the serum half-life of biological agents. Furthermore, PEGylated polypeptides are sometimes referred to as monoPEGylated, diPEGylated, trimonoPEGylated (etc.), because they refer to polypeptides in which one, two, three (or more) PEG moieties are attached to the polypeptide, respectively. In some embodiments, PEG may be attached to the sdAb directly or covalently (e.g., via a lysine side chain, a sulfhydryl group of cysteine, or an N-terminal amine), and optionally, a linker is used between the PEG and the sdAb. In some embodiments, the IL10Rb-binding molecule contains multiple PEG molecules, each of which is attached to a different amino acid residue. In some embodiments, the sdAb may be modified by incorporating a non-natural amino acid with a non-natural amino acid side chain to facilitate site-directed PEGylation. In other embodiments, one or more cysteine ​​residues at positions within sdAb may be substituted to facilitate site-directed PEGylation via cysteine ​​sulfhydryl side chains.

[0125] In some cases, the IL10Rb-binding molecules of this disclosure have an N-terminal glutamine ("1Q") residue. The N-terminal glutamine residue has been observed to spontaneously cyclize under or near physiological conditions to form a pyroglutamate (pE) (see, e.g., Liu, et al (2011) J. Biol. Chem. 286(13): 11211-11217). In some embodiments, the formation of a pyroglutamate complicates the N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. Consequently, when PEGylating the IL10Rb-binding molecules of this disclosure, particularly when aldehyde chemistry is used, position 1 of the IL10Rb-binding molecule having an amino acid (e.g., 1Q) at position 1 is replaced with an alternative amino acid or position 1 is deleted (e.g., des-1Q). In some embodiments, the IL10Rb binding molecule of this disclosure includes an amino acid substitution selected from the group of Q1E and Q1D.

[0126] PEGs suitable for conjugation into polypeptide sequences are generally soluble in water at room temperature and have the general formula: R(O-CH2-CH2) n Ure The formula has the following characteristics, where R is a hydrogen atom or a protecting group, such as an alkyl group or an alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has 1 to 8 carbon atoms. PEG may be linear or branched. Branched PEG derivatives, "star PEG", and multi-armed PEG are intended by this disclosure.

[0127] The molecular weight of PEG used in IL10Rb-binding molecules is not limited to any particular range. The molecular weight of the PEG component of the IL10Rb-binding molecule may be greater than approximately 5 kDa, greater than approximately 10 kDa, greater than approximately 15 kDa, greater than approximately 20 kDa, greater than approximately 30 kDa, greater than approximately 40 kDa, or greater than approximately 50 kDa. In some embodiments, the molecular weight is approximately 5 kDa to 10 kDa, approximately 5 kDa to 15 kDa, approximately 5 kDa to 20 kDa, approximately 10 kDa to 15 kDa, approximately 10 kDa to 20 kDa, approximately 10 kDa to 25 kDa, or approximately 10 kDa to 30 kDa. Linear or branched PEG molecules having a molecular weight of approximately 2,000 to 80,000 daltons, or approximately 2,000 to 70,000 daltons, or approximately 5,000 to 50,000 daltons, or approximately 10,000 to 50,000 daltons, or approximately 20,000 to 50,000 daltons, or approximately 30,000 to 50,000 daltons, or approximately 20,000 to 40,000 daltons, or approximately 30,000 to 40,000 daltons. In one embodiment of the IL10Rb binding molecule, the PEG is a 40kD branched PEG containing two 20kD arms.

[0128] This disclosure also intends IL10Rb-binding molecules comprising multiple PEG moieties, wherein the PEGs have different size values, and therefore various different PEGs are present in specific ratios. For example, in the preparation of PEGylated IL10Rb-binding molecules, some compositions include mixtures of monoPEGylated, diPEGylated, triPEGylated, and quadraPEGylated sdAb conjugates. In some compositions, the percentage of monoPEGylated species is 18–25%, the percentage of diPEGylated species is 50–66%, the percentage of triPEGylated species is 12–16%, and the percentage of quadraPEGylated species is up to 5%. Such complex compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to separate the conjugate fractions, and then, for example, a fraction containing conjugates with a desired number of PEGs attached is identified and purified from the unmodified protein sequence and the conjugates with other numbers of PEGs attached.

[0129] PEGylation most frequently occurs at the α-amino group at the N-terminus of polypeptides, the ε-amino group in the side chain of lysine residues, and the imidazole group in the side chain of histidine residues. Since most recombinant polypeptides have one α-amino group and numerous ε-amino and imidazole groups, a great many positional isomers can be produced depending on the linker chemistry.

[0130] Two widely used first-generation activated monomethoxyPEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence et al., U.S. Patent No. 5,650,234), which preferentially react with lysine residues to form carbamate bonds, but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site at the N-terminus of the polypeptide via reductive amination.

[0131] PEG can be bound to the IL10Rb binding molecule of this disclosure via terminal reactive groups ("spacers") that mediate the binding between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEG having spacers that can bind to free amino groups includes N-hydroxysuccinilimide polyethylene glycol, which can be prepared by activating a succinate ester of polyethylene glycol with N-hydroxysuccinilimide.

[0132] In some embodiments, PEGylation of sdAb is facilitated by incorporating a non-natural amino acid having a unique side chain that promotes site-specific PEGylation. It is known in the art that incorporating a non-natural amino acid into a polypeptide to provide a functional moiety is necessary to achieve such site-specific PEGylation of the polypeptide. For example, see Ptacin, et al., PCT international application number PCT / US2018 / 045257, filed on 3 August 2018 and published on 7 February 2019 under international publication number WO2019 / 028419Al.

[0133] The PEG portion of the PEGylated IL10Rb-binding molecule may be linear or branched. Branched PEG derivatives, "star PEG," and multi-armed PEG are intended by this disclosure.Specific embodiments of PEG useful in implementing this disclosure include 10 kDa linear PEG-aldehydes (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS esters (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehydes (e.g., Sunbright® ME-200AL, NOF), and 20 kDa linear PEG-NHS esters (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS). NOF), 20kDa 2-arm branched PEG-aldehyde, 20kDA PEG-aldehyde containing two 10kDA linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), 20kDa 2-arm branched PEG-NHS ester, 20kDA PEG-NHS ester containing two 10kDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40kDa 2-arm branched PEG-aldehyde, 40kDA PEG-aldehyde containing two 20kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3), 40kDa 2-arm branched PEG-NHS ester, 40kDA PEG-aldehyde containing two 20kDA linear PEG molecules This includes PEG-NHS esters (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30kDa PEG-aldehydes (e.g., Sunbright® ME-300AL), and linear 30kDa PEG-NHS esters.

[0134] Fc fusion In some embodiments, the carrier molecule is an Fc molecule or its monomeric subunit. In some embodiments, the dimeric Fc molecule may be manipulated to have a "knob-into-hole modification." Knob-into-hole modifications are further described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621, as well as in U.S. Patent No. 5,731,168 issued March 24, 1998, U.S. Patent No. 7,642,228 issued January 5, 2010, U.S. Patent No. 7,695,936 issued April 13, 2010, or U.S. Patent No. 8,216,805 issued July 10, 2012. Knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, where (i) an amino acid residue in the CH3 domain of the first heavy chain is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan), creating a protrusion ("knob") from the surface, and (ii) an amino acid residue in the CH3 domain of the second heavy chain is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby creating a cavity ("hole") inside the interface in the second CH3 domain, and the protruding side chain ("knob") of the first CH3 domain is accommodated in the cavity in the second CH3 domain. In one embodiment, the "knob-into-hole modification" includes the amino acid substitution T366W, optionally with the amino acid substitution S354C in one antibody heavy chain and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain may be modified by introducing cysteine ​​residues at positions S354 and Y349, resulting in the creation of a stabilizing disulfide bond between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15).The knob-into-hole configuration is used to facilitate the expression of a heterodimer polypeptide conjugate, specifically a first polypeptide (e.g., IL10Rb-bound sdAb) on a first Fc monomer having a "knob" modification, and a second polypeptide on a second Fc monomer having a "hole" modification.

[0135] Targeted domains In some embodiments, the IL10Rb-binding molecule is functionally linked to a targeting domain to facilitate selective binding to specific cell types or tissues that express cell surface molecules that specifically bind to such targeting domains, and optionally incorporates a linker between the IL10Rb-binding sdAb sequence of the fusion protein and the sequence of the targeting domain.

[0136] In some embodiments of IL10Rb-binding molecules, the IL10Rb-binding molecule can be targeted to specific cell types by incorporating a targeting domain into the structure of the IL10Rb-binding molecule. As used herein, the term targeting domain refers to a portion that specifically binds to a molecule expressed on the surface of the target cell. The targeting domain may be any portion that specifically binds to one or more cell surface molecules (e.g., T cell receptors) expressed on the surface of the target cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is an IL10Rb+ cell.

[0137] In some embodiments, the targeting domain is a ligand for a receptor. In some embodiments, the targeting domain is a ligand for a receptor expressed on the surface of a T cell. In some embodiments, the ligand is a cytokine. In some embodiments, cytokines include, but are not limited to, interleukins, interferons, and their functional derivatives. In some embodiments, cytokines include, but are not limited to, IL2, IL3, IL4, IL7, IL9, IL12, IL15, IL18, IL21, IL22, IL23, IL27, IL28, IL34, and modified versions or fragments thereof that bind to cognitive ligands expressed on the surface of a T cell. In some embodiments, cytokines include, but are not limited to, interferon α, interferon α2b, interferon γ, or interferon λ, and modified versions or fragments thereof that bind to cognitive ligands expressed on the surface of a T cell.

[0138] In another aspect, the present disclosure provides a polyvalent binding molecule comprising (a) an IL10Rb binding molecule and (b) a second binding molecule that specifically binds to the extracellular domain of a second cell surface molecule, wherein the IL10Rb binding molecule and the second binding molecule are functionally linked, optionally via a chemical linker or polypeptide linker. In some aspects, the IL10Rb binding molecule of the present disclosure is useful in the preparation of polyvalent binding molecules described in Gonzalez, et al. PCT / US2018 / 021301, published as WO2018 / 182935A1 on 4 October 2018. In some aspects, the second binding molecule specifically binds to (i) a cytokine receptor component that activates the cellular JAK / STAT pathway; (ii) a receptor tyrosine kinase; or (iii) the extracellular domain of a TNFR superfamily member. In some embodiments, the second surface molecule is a tyrosine kinase selected from EGFR, ErbB2, ErbB3, ErbB4, InsR, IGF1R, InsRR, PDGFRα, PDGFRβ, CSF1R / Fms, cKit, Flt-3 / Flk2, VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7 / CCK4, TrkA, TrkB, TrkC, Ror1, Ror2, MuSK, Met, Ron, Axl, Mer, Tyro3, ​​Tie1, Tie2, EphA1-8, EphA10, EphB1-4, EphB6, Ret, Ryk, DDR1, DDR2, Ros, LMR1, LMR2, LMR3, ALK, LTK, and SuRTK106 / STYK1.In some embodiments, the second surface molecule is TNFR1 (TNFRSF1A), TNFR2 (TNFRSF1B;TNFRSF2), 41-BB (TNFRSF9); AITR (TNFRSF18); BCMA (TNFRSF17), CD27 (TNFRSF7), CD30 (TNFRSF8), CD40 (TNFRSF5), death receptor 1 (TNFRSF10C), death receptor-3 (TNFRSF25), death receptor 4 (TNFRSF10A), death receptor 5 (TNFRSF10B), death receptor-6 (TNFRSF21), decoy receptor-3 (TNFRSF6B), decoy receptor 2 (TNFRSF10D), EDAR, Fas (TNFRSF6), HVEM (TNFRSF14). LTBR It is a TNFR superfamily member selected from (TNFRSF3), OX40 (TNFRSF4), RANK (TNFRSF11A), TACI (TNFRSF13B), Troy (TNFRSF19), XEDAR (TNFRSF27), Osteoportegerin (TNFRSF11B), TWEAK receptor (TNFRSF12A), BAFF receptor (TNFRSF13C), and NGF receptor (TNFRSF16).

[0139] In some embodiments, the targeting domain of the IL10Rb-binding molecule is an antibody (a molecule, as defined above, including molecules such as VHH and scFv). Examples of antibodies that can be incorporated as the targeting domain of the IL10Rb-binding molecule include, but are not limited to, anti-GD2 antibodies, anti-BCMA antibodies, anti-CD19 antibodies, anti-CD33 antibodies, anti-CD38 antibodies, anti-CD70 antibodies, anti-GD2 antibodies and IL3Ra2 antibodies, anti-CD19 antibodies, anti-mesothelin antibodies, anti-Her2 antibodies, anti-EpCam antibodies, anti-Muc1 antibodies, anti-ROR1 antibodies, anti-CD133 antibodies, anti-CEA antibodies, anti-PSMA antibodies, anti-EGRFRVIII antibodies, anti-PSCA antibodies, anti-GPC3 antibodies, anti-Pan-ErbB antibodies, and anti-FAP antibodies.

[0140] The aforementioned antibody or its antigen-binding fragment can be linked to another antibody to form, for example, a bispecific antibody or a multispecific antibody.

[0141] sign In some embodiments, the IL10Rb-binding molecule of this disclosure is functionally linked to a label. In some embodiments, the label is incorporated to facilitate use as an imaging agent, a diagnostic agent, or for use in cell sorting procedures. The term "label" includes, but is not limited to, fluorescent labels, biologically active enzyme labels, radioisotopes (e.g., radioactive ions), nuclear magnetic resonance-activated labels, luminescence labels, or magnetic compounds. In one embodiment, an IL10Rb-binding sdAb (e.g., IL10Rb-binding VHH) molecule stably associates (e.g., covalently, coordinately) with an imaging label. The term "imaging label" is used to describe any variety of compounds that are signatures that facilitate the identification, tracking, and / or measurement of the location of IL10Rb-binding sdAb (or its metabolites) using diagnostic procedures. Examples of imaging labels include, but are not limited to, fluorescent compounds, radioactive compounds, and compounds that do not pass through imaging methods (e.g., X-rays, ultrasound). Examples of radioactive compounds useful as imaging markers include technetium-99m( 99m Tc), Indium-111( 111 In), Iodine-131( 131 I) Iodine-123 123 I) Iodine-125 125 I), Gallium-67 ( 67 Ga), and Lutetium-177( 177 Lu), Lin ( 32 P), carbon ( 14 C), tritium ( 3 H), Yttrium ( 90 Y), Actinium ( 225 Ac), astatine ( 211 At), Rhenium ( 186 Re), bismuth ( 212 Bi or 213 Bi), and rhodium ( 188 This includes, but is not limited to, Rh.

[0142] Therapeutic drugs In some embodiments, the IL10Rb binding molecules of this disclosure are functionally linked to therapeutic agents. Examples of therapeutic agents include therapeutic small molecules (e.g., chemotherapeutic agents) or biotherapeutic agents, such as antibodies, cytotoxic or cell division-arresting compounds, radioisotopes, plant-derived, fungal or bacterial molecules, or biological proteins (e.g., protein toxins) or particles (e.g., nanoparticles or recombinant viral particles, e.g., recombinant viral particles via viral coating proteins), therapeutic antibodies, and chemotherapeutic agents, as will be further fully described herein.

[0143] In some embodiments, the therapeutic agent is a short-range radiation emitter functionally linked to the IL10Rb binding molecule of this disclosure, for example, a short-range, high-energy α- emitter. Examples of such radioisotopes include α- emitters, β- emitters, γ- emitters, or β / γ- emitters. Radioisotopes useful as therapeutic agents include yttrium-90( 90 Y), Lutetium-177( 177 Lu), Actinium-225 ( 225 Ac), Astatine-211 ( 211 at), Rhenium-186( 186 Re), Bismuth-212 212 Bi), Bismuth-213 213 Bi), and Rhodium-188( 188 It contains Rh.

[0144] Synthesis of IL10Rb binding molecules: In some embodiments, the IL10Rb-binding molecules of this disclosure are polypeptides. However, in some embodiments, only a portion of the IL10Rb-binding molecule is a polypeptide, for example, the IL10Rb-binding molecule includes a non-peptidyl domain (e.g., a PEG IL10Rb-binding sdAb conjugate, a radionucleotide IL10Rb-binding sdAb conjugate, or a small molecule IL10Rb-binding sdAb conjugate). The following provides guidance to enable solid-phase and recombinant synthesis of the polypeptide portion (domain) of the IL10Rb-binding molecules of this disclosure. In embodiments where only a portion of the IL10Rb-binding molecule is a polypeptide, the peptidyl domain of the IL10Rb-binding molecule will be understood as a process intermediate that may undergo further processing to complete the synthesis of the desired IL10Rb-binding molecule. The polypeptide domain of the IL10Rb-binding molecule may be produced by conventional methodologies for polypeptide construction, including recombinant or solid-phase synthesis, as described in more detail below.

[0145] chemical synthesis In addition to producing mutant polypeptides through the expression of nucleic acid molecules modified by recombinant molecular biology techniques, the polypeptide domain of IL10Rb-binding molecules can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those skilled in the art. Chemical synthesis includes direct peptide synthesis by chemical means of the polypeptide domain of IL10Rb-binding molecules exhibiting the described properties. This method allows for the incorporation of native and non-native amino acids at desirable positions that facilitate the linkage of specific molecules (e.g., PEG).

[0146] In some embodiments, the polypeptide domain of the IL10Rb-binding molecule of this disclosure can be produced by chemical synthesis. The chemical synthesis of the polypeptide domain of the IL10Rb-binding molecule may proceed via the liquid phase or via the solid phase. Using solid-phase peptide synthesis (SPPS) allows for the incorporation of non-natural amino acids and / or peptide / protein backbone modifications. Various types of SPPS can be used to synthesize the polypeptide domain of the IL10Rb-binding molecule of this disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, α-functional groups and any reactive side chains may be protected by acid-unstable or basic-acid-unstable groups that are stable under conditions for amide bond linking but can be easily cleaved without damaging the formed peptide chain.

[0147] In solid-phase synthesis, either the N-terminal or C-terminal amino acid can be bound to a suitable support material. A suitable support material is inert to the reagents and reaction conditions for the stepwise condensation and cleavage reactions of the synthesis process and does not dissolve in the reaction medium used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactive groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers, etc. Peptide synthesis can be carried out by sequentially binding protective amino acids according to conventional methods, typically in automated peptide synthesizers.

[0148] At the end of solid-phase synthesis, the peptide is cleaved from the support material, with the side-chain protecting groups being simultaneously cleaved. The resulting peptide can be purified by a variety of chromatographic methods, including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high-pressure liquid chromatography (HPLC), and reversed-phase HPLC.

[0149] Recombinant production Alternatively, the polypeptide domain of the IL10Rb-binding molecule of this disclosure may be produced by recombinant DNA technology. In a typical implementation of recombinant polypeptide production, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression is to be performed. This nucleic acid sequence is functionally ligated to one or more expression regulatory sequences encoded by the vector and functions in the target host cell. The recombinant protein may be recovered by disrupting the host cell, or it may be recovered from the cell medium if a secretion leader sequence (signal peptide) is incorporated into the polypeptide. The recombinant protein may be purified and concentrated for further use, including integration.

[0150] Synthesis of nucleic acid sequences encoding IL10Rb binding molecules In some embodiments, the polypeptide domain of the IL10Rb-binding molecule is produced by a recombinant method using a nucleic acid sequence encoding the polypeptide domain of the IL10Rb-binding molecule (or a fusion protein containing the polypeptide domain of the IL10Rb-binding molecule). The nucleic acid sequence encoding the desired polypeptide domain of the IL10Rb-binding molecule can be synthesized by chemical means using an oligonucleotide synthesizer.

[0151] Nucleic acid molecules are not limited to sequences that encode polypeptides. They may also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence of the polypeptide domain of an IL10Rb-binding molecule). Those skilled in molecular biology are familiar with routine procedures for isolating nucleic acid molecules. For example, nucleic acid molecules can be produced by treating genomic DNA with restriction endonucleases or by performing polymerase chain reactions (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0152] The nucleic acid molecules encoding the polypeptide domain (and its fusion) of the IL10Rb binding molecule may contain a native sequence, or a sequence different from the naturally occurring one, but which, due to genetic code degeneracy, encodes the same polypeptide. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, the nucleic acid molecules may be double-stranded or single-stranded (i.e., either a sense strand or an antisense strand).

[0153] The nucleic acid sequences encoding the polypeptide domain of the IL10Rb-binding molecule may be obtained from various commercial suppliers that provide custom synthesis of nucleic acid sequences. The amino acid sequence variants of the IL10Rb-binding molecule of this disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code well known in the art. Such variants are insertions, substitutions, and / or specified deletions of residues as mentioned herein. Any combination of insertions, substitutions, and / or specified deletions may be added to arrive at the final construct, provided that the final construct has the desired biological activity as defined herein.

[0154] Methods for constructing DNA sequences encoding the polypeptide domain of the IL10Rb-binding molecule, and for expressing these sequences in a properly transformed host, include, but are not limited to, the use of PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues to the polypeptide domain of the IL10Rb-binding molecule can also be introduced using standard recombination methods. In the case of deletions or additions, optionally, a nucleic acid molecule encoding the polypeptide domain of the IL10Rb-binding molecule is digested by a suitable restriction endonuclease. The resulting fragment may be expressed directly or further manipulated, for example, by ligation to a second fragment. Ligation may be facilitated if the two ends of the nucleic acid molecule contain complementary nucleotides that overlap each other, but blunt-ended fragments can also be ligated. Nucleic acids produced by PCR can also be used to create a variety of mutant sequences.

[0155] The polypeptide domain of the IL10Rb-binding molecule of this disclosure may be produced not only directly by recombination, but also as a fusion polypeptide with a heterologous polypeptide, such as a signal sequence, or another polypeptide having a specific cleavage site at the N-terminus or C-terminus of the mature IL10Rb-binding molecule. Generally, the signal sequence may be a component of the vector or part of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed by a host cell (i.e., cleaved by a signal peptidase). In some embodiments, the signal sequence is a signal sequence that is naturally associated with the IL10Rb-binding molecule (i.e., a human IL10Rb signal sequence). The incorporation of the signal sequence depends on whether it is desirable to secrete the IL10Rb-binding molecule from the recombinant cell from which the IL10Rb-binding molecule is produced. If the selected cell is a prokaryote, it is generally preferable that the DNA sequence does not encode the signal sequence. If the selected cell is a eukaryote, it is generally preferable that the signal sequence is encoded, and most preferably, a wild-type IL-2 signal sequence is used. Alternatively, signal sequences derived from secretory polypeptides of the same or related species, as well as heterogeneous mammalian signal sequences such as viral secretion leaders, e.g., the herpes simplex gD signal, may be suitable. If the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, an α-conjugation factor secretion signal sequence may be used to cause extracellular secretion of an IL10Rb-binding molecule into the culture medium, as described in Singh, U.S. Patent No. 7,198,919B1.

[0156] If the polypeptide domain of the IL10Rb-binding molecule to be expressed is to be expressed as a chimeric protein (e.g., a fusion protein containing the IL10Rb-binding molecule and a heterologous polypeptide sequence), the chimeric protein may be encoded by a hybrid nucleic acid molecule containing a first sequence encoding all or part of the polypeptide domain of the IL10Rb-binding molecule and a second sequence encoding all or part of the heterologous polypeptide. For example, the polypeptide domain of the IL10Rb-binding molecule described herein may be fused with a hexahistidine tag (SEQ ID NO: 170) to facilitate the purification of the protein expressed by bacteria, or with a hemagglutinin tag to facilitate the purification of the protein expressed in eukaryotic cells. The first and second should not be understood as limitations on the orientation of the elements of the fusion protein, as the heterologous polypeptide can be ligated to either the N-terminus and / or C-terminus of the polypeptide domain of the IL10Rb-binding molecule. For example, the N-terminus may be linked to a targeting domain, and the C-terminus may be linked to a chelated peptide purification handle (e.g., Hisx6 (SEQ ID NO: 170), Hisx8 (SEQ ID NO: 171)).

[0157] A back-translated gene can be constructed using the complete amino acid sequence of the polypeptide domain of the IL10Rb-binding molecule (or fusion / chimera) to be expressed. A DNA oligomer containing the nucleotide sequence encoding the polypeptide domain of the IL10Rb-binding molecule can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0158] In some embodiments, the nucleic acid sequence encoding the polypeptide domain of the IL10Rb binding molecule may be “codon-optimized” to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide variety of expression systems, including mammalian host cells, yeast host cells, and bacterial host cells, are well known in the art, and there are online tools for providing codon-optimized sequences for expression in various host cell types. For example, see Hawash, et al., (2017) 9:46-53, and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols See David Hacker (Human Press New York) for further information. Additionally, there are various web-based online software packages freely available to assist in the preparation of codon-optimized nucleic acid sequences.

[0159] Expression vector Once assembled (by synthesis, site-directed mutagenesis, or other means), the nucleic acid sequence encoding the polypeptide domain of the IL10Rb-binding molecule is inserted into an expression vector. Various expression vectors are available for use in different host cells and are typically based on the host cell for expression. An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, and embedded vectors. Plasmids are an example of a non-viral vector. To facilitate the efficient expression of recombinant polypeptides, the nucleic acid sequence encoding the polypeptide sequence to be expressed is functionally ligated to transcriptional and translational regulatory sequences that function in the selected expression host.

[0160] Expression vectors typically contain a selection gene, also known as a selection marker. This gene encodes a protein necessary for the survival or proliferation of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that compensate for nutritional deficiencies; or (c) proteins that supply essential nutrients not available from the complex medium.

[0161] The expression vectors for the polypeptide domain of the IL10Rb-binding molecule of this disclosure contain a regulatory sequence that is recognized by a host organism and functionally linked to a nucleic acid sequence encoding the polypeptide domain of the IL10Rb-binding molecule. The terms “regulatory sequence,” “regulatory sequence,” or “expression regulatory sequence” are used herein synonymously to refer to promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). For example, see Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, USA). Regulatory sequences include those that induce constitutive expression of nucleotide sequences in many types of host cells, and those that induce expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of expression vectors may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. In selecting an expression regulatory sequence, various factors understood by those skilled in the art must be considered. These include, for example, the relative strength of the sequence, its controllability, and, in particular, its compatibility with the actual DNA sequence encoding the IL10Rb binding molecule, with respect to its potential secondary structure.

[0162] In some embodiments, regulatory sequences are promoters, and promoters are selected, for example, based on the cell type to which expression is desired. A promoter is an untranslated sequence located upstream (5') of the start codon of a structural gene (typically within approximately 100–1000 bp) that controls the transcription and translation of a specific functionally linked nucleic acid sequence. Such promoters are typically divided into two classes: inductive promoters and constitutive promoters. Inductive promoters are those that initiate high levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or temperature changes. A large number of promoters recognized by various potential host cells are well known.

[0163] The T7 promoter can be used in bacteria, the polyhedrin promoter in insect cells, and the cytomegalovirus or metallothionein promoter in mammalian cells. Similarly, in higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are so named because of their ability to induce the expression of nucleic acid molecules in certain tissues or cell types within the body. Those skilled in the art are well aware of the many promoters and other regulatory elements that can be used to induce nucleic acid expression.

[0164] Transcription from a vector in mammalian host cells may be controlled by promoters obtained from the genomes of viruses, e.g., polyomavirus, fowlpox virus, adenovirus (e.g., human adenovirus serotype 5), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., mouse stem cell virus), hepatitis B virus, most preferably Simian virus 40 (SV40), heterozoan promoters, e.g., actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoter, or heat shock promoter, if such promoters are compatible with the host cell line. Conveniently, the early and late promoters of the SV40 virus can be obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication.

[0165] Transcription in higher eukaryotes is often increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting DNA elements of about 10–300 bp that act on promoters to increase transcription. Enhancers are relatively directional and position-independent and have been found to be located at the 5' and 3' ends of the transcription unit, within introns, and within the coding sequence itself. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, enhancers derived from eukaryotic viruses are commonly used. Examples include the SV40 enhancer located late at the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located late at the origin of replication, and the adenovirus enhancer. Enhancers may be spliced ​​and placed at the 5' or 3' end of the coding sequence in the expression vector, preferably located 5' from the promoter. Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences can generally be obtained from the 5' untranslated region, and sometimes the 3' untranslated region, of eukaryotic or viral DNA or cDNA. Standard techniques are used to construct appropriate vectors containing one or more of the components listed above.

[0166] In addition to sequences that facilitate the transcription of the inserted nucleic acid molecule, the vector may also contain other genes encoding replication origins and selection markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells expressing the neomycin resistance (neoR) gene, thus enabling phenotypic selection of transfected cells. Further examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can easily determine whether a particular regulatory element or selection marker is suitable for use in a particular experimental context. The correct assembly of an expression vector can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host.

[0167] host cell Furthermore, this disclosure provides prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding the polypeptide domain of an IL10Rb-binding molecule. The cells of this disclosure are transfected cells, i.e., cells into which a nucleic acid molecule, such as one encoding the polypeptide domain of an IL10Rb-binding molecule, has been introduced by recombinant DNA. Progeny of such cells are also considered to be within the scope of this disclosure.

[0168] Host cells are typically selected according to their compatibility with the chosen expression vector, the toxicity of the product encoded by the DNA sequence of this IL10Rb binding molecule, its secretory properties, its ability to correctly fold the polypeptide, its fermentation or culture requirements, and the ease of purifying the product encoded by the DNA sequence. Suitable host cells for cloning or expressing DNA in a vector as used herein are prokaryotes, yeasts, or higher eukaryotic cells.

[0169] In some embodiments, recombinant polypeptide domains of IL10Rb-binding molecules or their biologically active variants can also be produced in eukaryotes such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors usable for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, This includes pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

[0170] Examples of useful mammalian host cell lines include mouse L cells (LM[TK-], ATCC#CRL-2648), monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human fetal kidney cells (HEK293 cells or HEK293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR(CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); and human lung cells (W138, ATCC CCL 34). 75); human liver cells (Hep G2, HB 8065); mouse mammary gland tumors (MMT 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and human hepatome strain (HepG2). In mammalian cells, the regulatory function of expression vectors is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40.

[0171] The polypeptide domain of the IL10Rb-binding molecule may be produced in a prokaryotic host such as the bacterium Escherichia coli, or in a eukaryotic host such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH3T3 cells, or HeLa cells). These cells are available from many suppliers, including the American Type Culture Collection (Manassas, Va.). Those skilled in the art can make such a decision. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0172] In some embodiments, the recombinant polypeptide domain of the IL10Rb-binding molecule may or may not be glycosylated, depending on the host organism used to produce the IL10Rb-binding molecule. When bacteria are selected as the host, the polypeptide domain of the produced IL10Rb-binding molecule may be aglycosylated. On the other hand, eukaryotic cells may glycosylate the recombinant polypeptide domain of the IL10Rb-binding molecule.

[0173] For further expression systems in both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See also Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0174] Transfection The expression construct can be introduced into host cells to produce the recombinant polypeptide domain of the IL10Rb-binding molecule disclosed herein, or its biologically active mutein. The vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection methods. Appropriate methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0175] To facilitate the transfection of target cells, target cells may be directly exposed with a non-viral vector under conditions that facilitate the uptake of the non-viral vector. Examples of conditions that facilitate the uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt levels, and magnetic fields (electroporation).

[0176] cell culture Cells may be cultured in conventional nutrient media, which may be modified as appropriate for promoter induction, transformant selection, or amplification of genes encoding desired sequences. Mammalian host cells can be cultured in a variety of media. Suitable commercially available media for culturing host cells include Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma). To any of these media, hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or equivalent energy sources may be added as needed. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, have been previously used with host cells selected for expression and are apparent to those skilled in the art.

[0177] Recombinant protein recovery If a secretion leader sequence is used, the recombination-produced IL10Rb-binding polypeptide can be recovered from the culture medium as a secretion polypeptide. Alternatively, the IL10Rb-binding polypeptide can also be recovered from host cell lysates. During purification, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) may be used during the recovery from cell lysates to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants.

[0178] purification Various purification processes, such as affinity chromatography, are known and used in the art. Affinity chromatography typically utilizes highly specific binding sites present in biological macromolecules to separate molecules capable of binding to specific ligands. The ligand is covalently attached to an insoluble porous support medium in such a way that the ligand is explicitly presented on the protein sample, thereby separating and purifying a second species from the mixture using the innate specific binding of one molecular species. Antibodies are commonly used in affinity chromatography. Size selection processes may also be used to separate proteins according to their size, for example, gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography). In gel filtration, a protein solution packed with a semipermeable porous resin is passed through a column. The semipermeable resin has a range of pore sizes that determines the size of the proteins that can be separated by the column.

[0179] The recombinant polypeptide domain of the IL10Rb-binding molecule produced by the transformed host can be purified according to any suitable method. The IL10Rb-binding molecule may be isolated from inclusion bodies produced in E. coli by cation exchange, gel filtration, and / or reverse-phase liquid chromatography, or it may be isolated from conditional media derived from either a mammalian or yeast culture that produces a particular IL10Rb-binding molecule.

[0180] Recombinant polypeptides in substantially purified form can be used as therapeutic agents, for example, as described herein.

[0181] The biological activity of the recombinant polypeptide domain of the IL10Rb-binding molecule produced as described above was determined by competitive ELISA, radioligand binding assays (e.g., saturated binding, scatchard plot, non-linear curve fitting program, and competitive binding assay); non-radioligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009)) and commercially available measuring instruments from GE Healthcare Bio-Sciences, e.g., Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA) Confirmation can be made by IL10Rb binding using procedures well known in the art, including, but not limited to, liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays).

[0182] How to use Inhibition of IL10Rb receptor activity In one embodiment, the Disclosure provides a method for modulating the activity of IL10Rb-expressing cells by targeting and administering an IL10Rb-binding molecule in an amount sufficient to interfere with the activity of an IL10Rb-containing receptor. Furthermore, the Disclosure provides a method for modulating the activity of IL10Rb-expressing cells in a mixed cell population, comprising the step of contacting the cell population with the IL10Rb-binding molecule or complex of the Disclosure in vivo and / or ex vivo in an amount sufficient to interfere with the activity of an IL10Rb-containing receptor.

[0183] infectious disease In some embodiments, the compositions of this disclosure are useful as inhibitors of the IL-10 receptor, particularly through targeting of the IL10Rb subunit to downregulate the immunosuppressive effect of endogenous IL-10. The therapeutic and / or prophylactic activity of IL10R antagonists (e.g., IL10Rb-binding molecules) is well established in the scientific literature. For example, Von Herrath, et al. (U.S. Patent No. 7,553,932, issued June 30, 2009) disclose the use of IL10 receptor antagonist antibodies for the treatment of chronic acute infections, particularly chronic (persistent) or acute viral infections. Ejrnaes, et al. report on the use of anti-IL-10R antibodies as a demonstration of efficacy in an LCMV model of persistent viral infection. Ejrnaes, et al. (2006) J Exp Med 203(11):2461-2472. Kato, et al (U.S. Patent No. 8,420,784, issued April 16, 2011) describe that IL-10Rb inhibitory antibodies are useful in the treatment and prevention of pathogenic infections. Brooks, et al (J.Exp.Med.(2008)205(3)3:533-541;Nature Medicine(2001)12(11):1301-1309) describe that IL-10 receptor antagonists are useful in T cell recovery and prevention of persistent viral infections, and that blocking IL-10 activity enhances clearance of persistent viral infections.

[0184] In some embodiments, the compositions of the Disclosure may be administered in combination with one or more additional antiviral agents for the treatment of viral infections selected from, but not limited to, HIV, hepatitis C virus, hepatitis B virus, herpesvirus (HSV) types 1 and 2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and measles. In some embodiments, the compositions of the Disclosure may be administered in combination with vaccines for hepatitis C virus, hepatitis B virus, herpesvirus (HSV) types 1 and 2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and measles, and one or more antiviral agents selected from, but not limited to, acyclovir, ganciclovir, zidovudine (AZT), interferon-a2b, and interferon-a.

[0185] In some embodiments, the compositions of this disclosure may be used alone or in combination with one or more supplementary antibiotics in the treatment of bacterial infectious diseases. Examples of infectious diseases suitable for treatment with IL10 inhibitors include, but are not limited to, listeriosis (e.g., Listeria monocytogenes) (see, for example, Silva and Appleberg (2001) Antimicrobial Agents and Chemotherapy 45(4):1312-1314).

[0186] Autoimmune and inflammatory diseases In one embodiment, the Disclosure provides a method for treating a T cell-mediated autoimmune disease, comprising administering an IL10Rb-binding molecule in an effective amount to inhibit the T cell-mediated immune response. The IL10Rb-binding molecule of the Disclosure, alone or in association with other molecules, specifically binds to the ECD of IL10Rb and is useful in modulating the function of IL10Rb-expressing cells, and is useful in treating or preventing diseases, disorders, or conditions associated with inflammation or autoimmunity, where immunological memory is involved in the induction, maintenance, or exacerbation of diseases, disorders, or conditions. High serum levels of IL10 are associated with several autoimmune diseases, including but not limited to patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, Kawasaki disease, ulcerative colitis, Sjögren's syndrome, Graves' disease, myasthenia gravis, psoriasis, and autoimmune lymphoproliferative syndrome (ALPS). The compositions of this disclosure, as blockades of the competitive inhibitors IL-10 and the IL-10 receptor, are useful in the treatment of autoimmune diseases.

[0187] Disorders suitable for treatment with the IL10Rb binding molecules of this disclosure (including pharmaceutically acceptable formulations containing IL10Rb binding molecules, and / or nucleic acid molecules encoding such IL10Rb binding molecules, including recombinant viruses) include organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), and other conditions. Rohn's disease, diabetes including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegativity syndrome). This includes inflammatory or autoimmune diseases, including but not limited to ensopathy (arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteroarthritis, reactive arthritis, Reiter's syndrome, and SEA syndromes (seronegative, enthesopathy, and arthropathy syndromes).

[0188] Other examples of proliferation and / or differentiation disorders suitable for treatment with the IL10Rb binding molecules of this disclosure (including pharmaceutically acceptable formulations containing IL10Rb binding molecules, and / or encoding nucleic acid molecules including recombinant viruses encoding such IL10Rb binding molecules) include, but are not limited to, skin disorders. Skin disorders may involve abnormal activity of cells or groups of cells or layers in the skin, epithelium, or subcutaneous tissue layers, or abnormalities at the cutaneous-epithelial junction. For example, skin disorders may involve abnormal activity of keratinocytes (e.g., hyperproliferative basal and superbasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more of the epithelial layers, such as the basal layer (germinal layer), spinous layer, granular layer, slender layer, or stratum corneum. In other embodiments, the disorder may be accompanied by abnormal activity of skin cells, such as cutaneous endothelium, fibroblasts, or immune cells (e.g., mast cells or macrophages) found in skin layers, such as the papillary layer or reticular layer.

[0189] Examples of inflammatory or autoimmune skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), such as exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosacea, parapsoriasis, pityriasis lichenoides, lichen planus, lichen patella, and ichthyosiform dermatosis. Skin disorders include dermatosis, keratosis, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, bullous pemphigoid (e.g., ocular scarring pemphigoid or bullous pemphigoid), urticaria, porokeratosis, rheumatoid arthritis with hyperproliferation and inflammation of epithelial-associated intracellular wall joint capsules; dermatitis, e.g., seborrheic dermatitis and photodermatitis; keratosis, e.g., seborrheic keratosis, senile keratosis, actinic keratosis, and follicular keratosis; acne vulgaris; keloids and prevention of keloid formation; nevi; warts, condyloma or genital warts, and human papillomavirus (HPV) infections, e.g., warts including sexually transmitted warts; vitiligo; lichen planus; and keratitis. Skin disorders may include dermatitis, e.g., atopic dermatitis or allergic dermatitis, or psoriasis.

[0190] The compositions of this disclosure (including pharmaceutically acceptable formulations containing an IL10Rb binding molecule and / or encoding nucleic acid molecules, including recombinant viruses encoding such an IL10Rb binding molecule) may also be administered to patients who have (or may have) psoriasis or psoriatic disorders. The term “psoriasis” is intended to have its medical meaning, namely, a disease affecting primarily the skin and producing raised, thickened, desquamating, non-scarring lesions. The lesions are typically sharply demarcated erythematous papules covered with overlapping, shiny scales. The scales are typically silvery or slightly milky. Nail involvement frequently occurs, resulting in pitting, nail separation, thickening, and discoloration. Psoriasis may be associated with arthritis and can be limb-disabling. Keratinocyte hyperplasia, along with epithelial inflammation and reduced keratinocyte differentiation, is a key feature of psoriatic epithelial hyperplasia. Multiple mechanisms have been proposed to explain the keratinocyte hyperproliferation that characterizes psoriasis. Impaired cellular immunity has also been linked to the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic constant psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, psoriasis vulgaris, exanthematous psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or focal pustular psoriasis.

[0191] Combination with supplemental therapeutic agents This disclosure provides the use of the IL10Rb binding molecule of this disclosure in combination with one or more additional activators ("supplementary agents"). Such further combinations are interchangeably referred to as "supplementary combinations" or "supplementary combination therapies," and therapeutic agents used in combination with the IL10Rb binding molecule of this disclosure are referred to as "supplementary agents." As used herein, the term "supplementary agent" includes agents that can be administered or introduced separately, for example, in separate doses (for example, they may be provided in a kit) and / or that can be formulated separately for therapies that can be administered or introduced in combination with the IL10Rb binding molecule.

[0192] Where used herein, the term “in combination with” in reference to the administration of multiple agents to a subject means the administration of a first agent with at least one additional agent (i.e., a second, third, fourth, fifth, etc.) to the subject. For the purposes of the present invention, one agent (e.g., an IL10Rb binding molecule) is considered to be administered in combination with a second agent (e.g., an immune checkpoint pathway modulator) such that the therapeutic effects of the first and second agents overlap, and the biological effect resulting from the administration of the first agent persists in the subject at the time of administration of the second agent. For example, while the IL10Rb binding molecule of this disclosure is typically administered more frequently, e.g., daily, by BID, or weekly, a PD1 immune checkpoint inhibitor (e.g., nivolumab or pembrolizumab) is typically administered every two or three weeks by IV infusion. However, the administration of the first agent (e.g., pembrolizumab) provides a therapeutic effect over a long period, and the administration of the second agent (e.g., an IL10Rb binding molecule) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing. As a result, the second agent is considered to be administered in combination with the first agent, even if the first agent is administered at a point significantly later (e.g., days or weeks) from the time of administration of the second agent. In one embodiment, one agent is considered to be administered in combination with the second agent when the first and second agents are administered simultaneously (within 30 minutes of each other), synchronously, or sequentially. In some embodiments, the first agent is considered to be administered in the "same period" as the second agent when the first and second agents are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other. The term “in combination with” is also understood to apply to situations in which the first and second agents are co-formulated into a single pharmaceutically acceptable formulation, and the co-formulation is administered to the subject. In certain embodiments, the IL10Rb binding molecule and the supplementary agent are administered or applied sequentially, for example, one agent is administered before one or more other agents.In other embodiments, the IL10Rb binding molecule and the supplementary agent are administered simultaneously, for example, two or more agents are administered at the same or approximately the same time; two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0193] Adjunctive agents useful in the treatment of inflammatory or autoimmune disorders In some embodiments, the method further comprises administering the IL10Rb-binding molecule of the present disclosure in combination with one or more supplementary agents selected from the group consisting of corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines, and therapeutic antibodies. In certain embodiments, the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BlyS, CD11a, CD20, CD25, CD3, CD52, IgEIL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

[0194] In some embodiments, the supplementary agent is one or more agents selected from the group consisting of corticosteroids (including, but not limited to, prednisone, budesonide, and prednisone), Janus kinase inhibitors (including, but not limited to, tofacitinib (Xeljanz®)), calcineurin inhibitors (including, but not limited to, cyclosporine and tacrolimus), mTor inhibitors (including, but not limited to, sirolimus and everolimus), IMDH inhibitors (including, but not limited to, azathioprine, leflunomide, and mycophenolate), biologics such as abatacept (Orencia®) or etanercept (Enbrel®), and therapeutic antibodies.

[0195] Examples of therapeutic antibodies that can be administered as supplemental agents in combination with the IL10Rb binding molecules of this disclosure in the treatment of autoimmune diseases include anti-CD25 antibodies (e.g., daclizumab and basiliximab), anti-VLA-4 antibodies (e.g., natalizumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-CD20 antibodies (e.g., rituximab, ocrelizumab), anti-TNF antibodies (e.g., infliximab and adalimumab), anti-IL6R antibodies (e.g., tocilizumab), anti-TNFα antibodies (e.g., adalimumab (Humira®), golimumab, and i This includes, but is not limited to, nfliximab, anti-integrin-α4β7 antibodies (e.g., vedolizumab), anti-IL17a antibodies (e.g., brodalumab or secukinumab), anti-IL4Rα antibodies (e.g., dupilumab), anti-RANKL antibodies, IL6R antibodies, anti-IL1β antibodies (e.g., canakinumab), anti-CD11a antibodies (e.g., efalizumab), anti-CD3 antibodies (e.g., muromonab), anti-IL5 antibodies (e.g., mepolizumab, reslizumab), anti-BLyS antibodies (e.g., belimumab), and anti-IL12 / IL23 antibodies (e.g., ustekinumab).

[0196] Many therapeutic antibodies have been approved for clinical use for autoimmune diseases. Examples of antibodies approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of autoimmune diseases in subjects suffering from autoimmune diseases, which may be administered as supplemental agents (and optionally additional supplemental agents) in combination with the IL10Rb binding molecules of this disclosure for the treatment of indicated autoimmune diseases include atezolizumab, olaratumumab, ixekizumab, trastuzumab, infliximab, rituximab, edrecolomab, daratumumab, elotuzumab, nesitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, pertuzumab, brentuximab vedotin, and ipi This includes limumab, ofatumumab, certolizumab-pegol, catumakisomab, panitumumab, bevacizumab, ramucirumab, siltuximab, enfortumab-vedotin, polatuzumab-vedotin, [fam]-trastuzumab-deruxtecan, semiprimab, moxetumomab-pasudotox, mogamulizumab, tildrakizumab, ivalizumab, durvalumab, inotuzumab, ozogamicin, avelumab, obinutuzumab, adtrastuzumab emtansine, cetuximab, tositumomab-I131, ibritumomab tiuxetan, gemtuzumab, and ozogamicin. More antibodies useful as supplemental agents in carrying out the methods of this disclosure may be administered alone or in the form of any antibody-drug conjugate (ADC) comprising an antibody, a linker, and one or more drugs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 drugs), or in a modified form (e.g., PEGylated).

[0197] Treatment of neoplasms This disclosure provides a method for using IL10Rb-binding molecules in the treatment of subjects suffering from neoplasms, disorders, or conditions by administering a therapeutically effective amount of the IL10Rb-binding molecules described herein (or nucleic acids encoding IL10Rb-binding molecules, including recombinant vectors encoding IL10Rb-binding molecules, as well as eukaryotic and prokaryotic cells modified to express IL10Rb-binding molecules).

[0198] Neoplasms suitable for treatment: The compositions and methods of the present disclosure are useful in the treatment of subjects suffering from neoplastic diseases characterized by the presence of neoplasms, including benign and malignant neoplasms, and neoplastic diseases.

[0199] Examples of benign neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, adenomas, fibromas, hemangiomas, and lipomas. Examples of premalignant neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, carcinomas (cancers arising from epithelial tissues, such as the skin or tissues lining internal organs), leukemias, lymphomas, and sarcomas typically derived from bone, fat, muscle, blood vessels or connective tissues. Also included within the term neoplasm are virus-induced neoplasms such as warts and EBV-induced diseases (i.e., infectious mononucleosis), scar formation, intimal smooth muscle cell hyperplasia, restenosis, and hyperproliferative vascular diseases including vascular occlusion.

[0200] The term "neoplastic disease" refers to cancers characterized by solid tumors and non-solid tumors, including but not limited to, for example, breast cancer; sarcomas (including but not limited to osteosarcoma, angiosarcoma and fibrosarcoma), leukemias, lymphomas, genitourinary cancers (including but not limited to ovarian, urethral, bladder, and prostate cancers); gastrointestinal cancers (including but not limited to colorectal, esophageal, and gastric cancers); lung cancer; myeloma; pancreatic cancer; liver cancer; kidney cancer; endocrine cancers; skin cancers; and brain or central and peripheral nervous system (CNS) tumors, malignant or benign, such as gliomas and neuroblastomas, astrocytomas, myelodysplastic syndromes; cervical carcinoma-in-situ; intestinal polyposis; oral leukoplakia; histiocytosis, hypertrophic scars including keloid scars, hemangiomas; hyperplastic arteriolar stenosis, psoriasis, inflammatory arthritis; papulosquamous eruptions including hyperkeratosis and arthritis.

[0201] The term "neoplastic disease" includes carcinomas. The term "carcinoma" refers to a malignant tumor of epithelial or endocrine tissue, including carcinomas of the respiratory system, gastrointestinal system, urogenital system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. The term "neoplastic disease" includes adenocarcinomas. "Adenocarcinoma" refers to a carcinoma that is derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0202] As used herein, the term "hematopoietic neoplastic disorder" refers to a neoplastic disease with hyperplastic / neoplastic cells that originate from hematopoietic sources, such as the bone marrow, lymphoid or erythroid lineages, or their progenitor cells.

[0203] Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of undetermined lineage. Exemplary myeloid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML).

[0204] Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin lymphoma, and immunodeficiency-related lymphoproliferative disorders. Exemplary lymphoid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL) including B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenström macroglobulinemia (WM).

[0205] In some cases, hematopoietic neoplasms result from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term “hematopoietic neoplasm” refers to malignant lymphomas, including but not limited to non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin’s disease, and Reed-Sternberg’s disease.

[0206] The determination of whether a subject has a "neoplasmic disease" refers to a physician's determination of whether a subject requires or would benefit from treatment, based on acceptable available information in the field of disease, disorder, or condition identification, including but not limited to X-ray, CT scan, conventional laboratory diagnostic tests (e.g., blood cell count), genomic data, protein expression data, and immunohistochemistry.

[0207] Combinations of IL10Rb binding molecules with supplemental antineoplastic agents: This disclosure provides the use of the IL10Rb conjugate molecule of this disclosure in combination with one or more additional active antineoplastic agents ("complementary agents") for the treatment of neoplastic diseases. Such further combinations are interchangeably referred to as "complementary antineoplastic combinations" or "complementary antineoplastic combination therapies," and the therapeutic agents used in combination with the IL10Rb conjugate molecule of this disclosure are referred to as "complementary antineoplastic agents." As used herein, the term "complementary antineoplastic agent" includes antineoplastic agents that can be administered or introduced separately, for example, in separate doses (for example, provided in a kit) and / or formulated separately for therapies that can be administered or introduced in combination with the IL10Rb conjugate molecule.

[0208] Chemotherapy agents: In some embodiments, the supplementary antineoplastic agent is a chemotherapeutic agent. In some embodiments, the supplementary agent is a “cocktail” of multiple chemotherapeutic agents. In some embodiments, the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e.g., radiotherapy). The term “chemotherapeutic agent” includes alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and piposulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, including ethyleneimines and methylamines. Thyramelamine; Nitrogen mustard, e.g., chlorambucil, chlornafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenesterine, prednimustine, trophosphamide, uracil mustard; Nitrosourea, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine Antibiotics, e.g., acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, e.g., bleomycin A2, kactinomycin, calicheamicin, carabicin, kaminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin and derivatives, e.g., demethoxy-daunorubicin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxo Rubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, N-methylmitomycin C; mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, dideazatetrahydrofolate, and folic acid; 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, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisanthren; edatrexate; dehofamine; demecolsin; diazicon; elformitin; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluzone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; podophyllite 2-ethylhydrazide; procarbazine; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel, nab-paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate Platinum and platinum-coordinate complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; taxanes, e.g., paclitaxel, docetaxel, cabazitaxel;Carminomycin, adriamycin, e.g., 4'-epiaadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; colchicine and any pharmaceutically acceptable salts, acids, or derivatives thereof, including but not limited to these.

[0209] The term “chemotherapeutic agent” also includes anti-hormone agents that act to modulate or inhibit hormonal effects on tumors, which include, for example, anti-estrogens such as tamoxifen, raloxifen, 4(5)-imidazole inhibitory aromatase, 4-hydroxytamoxifen, trioxyfen, keoxyfen, onapristone, and toremifene; as well as anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0210] In some embodiments, supplementary antineoplastic agents include cytokines or cytokine antagonists, e.g., IL-12, INFα, or anti-epidermal growth factor receptors, irinotecan; tetrahydrofolate antagonists, e.g., pemetrexed; antibodies against tumor antigens, monoclonal antibody-toxin complexes, T-cell adjuvants, bone marrow grafts, or antigen-presenting cells (e.g., dendritic cell therapy); antitumor vaccines, replication-competent viruses, signaling inhibitors (e.g., Gleevec® or Herceptin®); or immunomodulators, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade). In addition to (registered trademark) and Enbrel (registered trademark), interferon-β1a (Avonex (registered trademark)), and interferon-β1b (Betaseron (registered trademark)), one or more chemical or biological agents identified in the art as useful in the treatment of neoplasms, including but not limited to one or more combinations of known chemotherapy regimens, including but not limited to TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily understood by clinicians skilled in the art.

[0211] In some embodiments, the IL10Rb-binding molecule is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor such as sunitinib, a PARP inhibitor such as olaparib, an EGFR inhibitor such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor such as epacadostat, and an oncolytic virus such as tarimozine-laharpalepbec (T-VEC).

[0212] Antitumor antigen antibody therapy as a supplementary agent In some embodiments, “complementary antineoplastic agents” are therapeutic antibodies (including, but not limited to, bispecific and trispecific antibodies that bind to one or more tumor-associated antigens, including bispecific T-cell engagers (BITEs), biaffinity retargeting (DART) constructs, and trispecific killer engagers (TriKE) constructs).

[0213] In some embodiments, the therapeutic antibody is HER2 (e.g., trastuzumab, pertuzumab, adtrastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilimumab), CD22 (e.g., moxetumomab pasudotox), CCR4 (e.g., mogamulizumab), IL23p19 (e.g., tildrakizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab), CD38 (e.g., daratumumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate-binding protein, GD2 (e.g., dinutuximab), GD3, IL6 (e.g., siltuximab), GM2, Le y The antibody is an antibody that binds to at least one tumor antigen selected from the group consisting of VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRa (e.g., ofatumumab), EGFR (e.g., cetuximab, panitumumab, and nesitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL R1, TRAIL R2, RANKL RAP, tenascin, integrin αVβ3, and integrin α4β1.

[0214] In some embodiments, therapeutic antibodies are immune checkpoint modulators for the treatment and / or prevention of diseases, disorders, or conditions associated with neoplasms, in addition to the neoplasm in the subject. The term “immune checkpoint pathway” refers to a biological response triggered by the binding of a first molecule (e.g., protein, e.g., PD1) expressed on an antigen-presenting cell (APC) to a second molecule (e.g., protein, e.g., PDL1) expressed on an immune cell (e.g., T cell) that modulates the immune response, either through stimulation of the immune response (e.g., upregulation of T cell activity) or inhibition of the immune response (e.g., downregulation of T cell activity). Molecules involved in the formation of binding pairs that modulate the immune response are generally referred to as “immune checkpoints.” In one embodiment, an immune checkpoint pathway modulator is a negative immune checkpoint pathway antagonist (“PD1 pathway inhibitor”) that inhibits the binding of PD1 to PDL1 and / or PDL2. The term PD1 pathway inhibitor includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Examples of commercially available PD1 pathway inhibitors useful as supplemental agents in the treatment of neoplasms include, but are not limited to, antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2, such as nivolumab (Opdivo®, BMS-936558, MDX1106, BristolMyers Squibb, commercially available from Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco CA).Additional PD1 pathway inhibitor antibodies are in clinical development and include, but are not limited to, durvalumab (MEDI4736, Medimmune / AstraZeneca), pizilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers Squibb), and avelumab (MSB0010718C, Merck Serono / Pfizer) and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149 (Genentech, Inc.), issued on July 10, 2012; U.S. Patent No. 8,168,757 (Merck Sharp and Dohme Corp.), issued on May 1, 2012; U.S. Patent No. 8,008,449 (Medarex), issued on August 30, 2011; and U.S. Patent No. 7,943,743 (Medarex, Inc.), issued on May 17, 2011.

[0215] Examples of FDA-approved antibody therapies that can be used as supplemental agents for the treatment of neoplasms include atezolizumab, olaratumab, ixekizumab, trastuzumab, infliximab, rituximab, edrecolomab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, pertuzumab, brentuximab vedotin, ipilimumab, ofatumumab, certolizumab-pegol, catumakisomab, panitumumab, and vegetumab. Includes bacizumab, ramucirumab, siltuximab, enfortumab vedotin, polatuzumab vedotin, [fam]-trastuzumab deruxtecan, semiprimab, moxetumomab pasdotox, mogamulizumab, tildrakizumab, ibalizumab, durvalumab, inotuzumab, ozogamicin, avelumab, obinutuzumab, adtrastuzumab emtansine, cetuximab, tositumomab-I131, ibritumomab tiuxetan, gemtuzumab, and ozogamicin.

[0216] physical method In some embodiments, the complementary antineoplastic agent is one or more non-pharmacological modalities (e.g., localized radiotherapy or total body radiotherapy or surgery). As an example, the disclosure envisions a treatment regimen in which the radiotherapy phase is preceded or followed by treatment in a treatment regimen comprising an IL10Rb-binding molecule and one or more complementary antineoplastic agents. In some embodiments, the disclosure further envisions the use of the IL10Rb-binding molecule in combination with surgery (e.g., tumor resection). In some embodiments, the disclosure further envisions the use of the IL10Rb-binding molecule in combination with bone marrow transplantation, peripheral blood stem cell transplantation or other types of transplantation therapy.

[0217] cell therapy In some embodiments, the methods of the present disclosure may involve a combination of administration of an IL10Rb-binding molecule with a supplementary agent in the form of a cell therapy for the treatment of neobiotic, autoimmune, or inflammatory diseases. Examples of cell therapies suitable for use in combination with the methods of the present disclosure include, but are not limited to, engineered T cell products containing one or more activated CAR-T cells, engineered TCR cells, tumor-infiltrating lymphocytes (TILs), and engineered Treg cells.

[0218] CAR-T useful in carrying out the present invention is prepared according to principles well known in the art. See, for example, Eshhaar et al., U.S. Patent No. 7,741,465 B1, published June 22, 2010; Sadelain, et al (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS (USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products include axicaptadine silolucel (marketed as Yescarta® and commercially available from Gilead Pharmaceuticals) and tisagenlecleucel (marketed as Kymriah® and commercially available from Novartis). In some embodiments, the CAR-T has a CAR that specifically binds to cell surface molecules associated with tumor cells, selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3R□2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP.

[0219] Identification, isolation, concentration, or depletion of IL10Rb+ cells In one embodiment, the Disclosure provides a method for using the IL10Rb-binding molecule of the Disclosure useful in a method for isolating, concentrating, or depleting IL10Rb+ cells from a biological sample containing IL10Rb+ cells. The biological sample may include T cells and B cells of hematopoietic origin, such as PBMCs, cell culture origin, or tissue origin, such as brain or bone marrow. Suitable methods for isolating, concentrating, or depleting IL10Rb+ cells include centrifugation, filtration, magnetic cell sorting, and fluorescent cell sorting by techniques well known in the Art. The Disclosure further provides a method for treating subjects suffering from disease, disorder, or condition by administering a therapeutically effective amount of cell product from which IL10Rb+ cells have been concentrated or depleted through the use of the IL10Rb-binding molecule described herein.

[0220] In one embodiment, the sorting procedure uses an IL10Rb-binding molecule containing a fluorescent label for use in FACS isolation or depletion of IL10Rb+ cells from a sample. The fluorescent label may be attached to the sdAb of the IL10Rb-binding molecule directly (e.g., by chemical conjugation using an optional linker) or indirectly (e.g., by biotinylation of the sdAb and binding of the biotinylated antibody to a streptavidin fluorescent dye conjugate). Such fluorescently labeled IL10Rb+ cells may be isolated from a mixed cell population using conventional FACS techniques.

[0221] In an alternative embodiment, the selection procedure uses an IL10Rb binding molecule of the present disclosure (e.g., an IL10Rb binding VHH) conjugated to magnetic particles that provide magnetic labeling of IL10Rb+ cells for use in a magnetic cell separation procedure. In one embodiment, the method comprises: (a) conjugating one or more IL10Rb binding molecules of the present disclosure (e.g., an IL10Rb binding VHH) to magnetic particles; (b) creating a mixture by contacting a biological sample with the magnetic particles conjugated to an amount of the IL10Rb binding molecule; (c) subjecting to a magnetic field such that magnetically labeled IL10Rb+ cells are retained; (d) removing non-magnetically labeled cells from the mixture; and (e) removing the magnetic field to enable isolation of the IL10Rb+ cells.

[0222] Cell selection procedures (e.g., FACS or magnetic separation) result in two products: (a) a population of cells depleted of IL10Rb+ cells and (b) a population of cells enriched in IL10Rb+ cells. Each of these populations may be further processed by conventional procedures to identify specific IL10Rb+ or IL10Rb− cell subsets that may be useful in research, diagnostic or clinical applications. For example, isolation of specific IL10Rb+ T cell subsets that also express one or more of CD4, CD8, CD19, CD25, and CD62L may involve further iterations using one or more antibodies that specifically bind to the CD4, CD8, CD19, CD25, and CD62L antigens by FACS or magnetic field separation using techniques well known in the art.

[0223] Combination with supplementary therapeutic agents This disclosure provides the use of the IL10Rb binding molecule of this disclosure in combination with one or more additional activators ("supplementary agents"). Such further combinations are interchangeably referred to as "supplementary combinations" or "supplementary combination therapies," and therapeutic agents used in combination with the IL10Rb binding molecule of this disclosure are referred to as "supplementary agents." As used herein, the term "supplementary agent" includes agents that can be administered or introduced separately, for example, in separate doses (for example, they may be provided in a kit) and / or that can be formulated separately for therapies that can be administered or introduced in combination with the IL10Rb binding molecule.

[0224] Where used herein, the term “in combination with” in reference to the administration of multiple agents to a subject means the administration of a first agent with at least one additional agent (i.e., a second, third, fourth, fifth, etc.) to the subject. For the purposes of the present invention, one agent (e.g., an IL10Rb binding molecule) is considered to be administered in combination with a second agent (e.g., an immune checkpoint pathway modulator) such that the therapeutic effects of the first and second agents overlap, and the biological effect resulting from the administration of the first agent persists in the subject at the time of administration of the second agent. For example, while the IL10Rb binding molecule of this disclosure is typically administered more frequently, e.g., daily, by BID, or weekly, a PD1 immune checkpoint inhibitor (e.g., nivolumab or pembrolizumab) is typically administered every two or three weeks by IV infusion. However, the administration of the first agent (e.g., pembrolizumab) provides a therapeutic effect over a long period, and the administration of the second agent (e.g., an IL10Rb binding molecule) provides its therapeutic effect while the therapeutic effect of the first agent is still ongoing. As a result, the second agent is considered to be administered in combination with the first agent, even if the first agent is administered at a point significantly later (e.g., days or weeks) from the time of administration of the second agent. In one embodiment, one agent is considered to be administered in combination with the second agent when the first and second agents are administered simultaneously (within 30 minutes of each other), synchronously, or sequentially. In some embodiments, the first agent is considered to be administered in the "same period" as the second agent when the first and second agents are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other. The term “in combination with” is also understood to apply to situations in which the first and second agents are co-formulated into a single pharmaceutically acceptable formulation, and the co-formulation is administered to the subject. In certain embodiments, the IL10Rb binding molecule and the supplementary agent are administered or applied sequentially, for example, one agent is administered before one or more other agents.In other embodiments, the IL10Rb binding molecule and the supplementary agent are administered simultaneously, for example, two or more agents are administered at the same or approximately the same time; two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0225] Chemotherapy agents In some embodiments, particularly in the treatment of neoplasms, the supplementary agent is a chemotherapeutic agent. In some embodiments, the supplementary agent is a “cocktail” of multiple chemotherapeutic agents. In some embodiments, the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e.g., radiotherapy). The term “chemotherapeutic agent” includes alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and piposulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, including ethyleneimines and methylmelamine. Thyramelamine; Nitrogen mustard, e.g., chlorambucil, chlornafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenesterine, prednimustine, trophosphamide, uracil mustard; Nitrosourea, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine Antibiotics, e.g., acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, e.g., bleomycin A2, kactinomycin, calicheamicin, carabicin, kaminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin and derivatives, e.g., demethoxy-daunorubicin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxo Rubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, N-methylmitomycin C; mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, dideazatetrahydrofolate, and folic acid; 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, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisanthren; edatrexate; dehofamine; demecolsin; diazicon; elformitin; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluzone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; podophyllite 2-ethylhydrazide; procarbazine; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel, nab-paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate Platinum and platinum-coordinate complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; taxanes, e.g., paclitaxel, docetaxel, cabazitaxel;Carminomycin, adriamycin, e.g., 4'-epiaadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; colchicine; and any pharmaceutically acceptable salts, acids, or derivatives thereof, including but not limited to these.

[0226] The term “chemotherapeutic agent” also includes anti-hormone agents that act to modulate or inhibit hormonal effects on tumors, which include, for example, anti-estrogens such as tamoxifen, raloxifen, 4(5)-imidazole inhibitory aromatase, 4-hydroxytamoxifen, trioxyfen, keoxyfen, onapristone, and toremifene; as well as anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0227] In some embodiments, supplementary agents include cytokines or cytokine antagonists, e.g., IL-12, INFα, or anti-epidermal growth factor receptors, irinotecan; tetrahydrofolate antagonists, e.g., pemetrexed; antibodies against tumor antigens, monoclonal antibody-toxin complexes, T-cell adjuvants, bone marrow grafts, or antigen-presenting cells (e.g., dendritic cell therapy); antitumor vaccines, replication-competent viruses, signaling inhibitors (e.g., Gleevec® or Herceptin®); or immunomodulators, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade®) to achieve additional or synergistic suppression of tumor growth. In addition to registered trademarks (Avonex®) and Enbrel®, interferon-β1a (Avonex®), and interferon-β1b (Betaseron®), one or more chemical or biological agents identified in the art as useful in the treatment of neoplasms, including but not limited to one or more combinations of known chemotherapy regimens, including but not limited to TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily understood by clinicians skilled in the art.

[0228] In some embodiments, the IL10Rb-binding molecule is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor such as sunitinib, a PARP inhibitor such as olaparib, an EGFR inhibitor such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor such as epacadostat, and an oncolytic virus such as tarimozine-laharpalepbec (T-VEC).

[0229] therapeutic antibodies In some embodiments, the “supplementary agent” is a therapeutic antibody (including, but not limited to, bispecific and trispecific antibodies that bind to one or more tumor-associated antigens, such as bispecific T-cell engagers (BITEs), biaffinity retargeting (DART) constructs, and trispecific killer engagers (TriKE) constructs).

[0230] In some embodiments, the therapeutic antibody is HER2 (e.g., trastuzumab, pertuzumab, adtrastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilimumab), CD22 (e.g., moxetumomab pasudotox), CCR4 (e.g., mogamulizumab), IL23p19 (e.g., tildrakizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab), CD38 (e.g., daratumumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate-binding protein, GD2 (e.g., dinutuximab), GD3, IL6 (e.g., siltuximab), GM2, Le y The antibody is an antibody that binds to at least one tumor antigen selected from the group consisting of VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFR (e.g., ofatumumab), EGFR (e.g., cetuximab, panitumumab, and nesitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL R1, TRAIL R2, RANKL RAP, tenascin, integrin αVβ3, and integrin α4β1.

[0231] Cell therapy agents and methods as supplementary agents In some embodiments, the methods of the present disclosure may include the administration of the IL10Rb-binding molecule of the present disclosure in combination with a supplementary agent in the form of a cell therapy for the treatment of neobiotic, autoimmune, or inflammatory diseases. Examples of cell therapies suitable for use in combination with the methods of the present disclosure include, but are not limited to, engineered T cell products including one or more first, second, third, or fourth generation CAR-T cells, engineered TCR cells, tumor-infiltrating lymphocytes (TILs), and engineered Treg cells. In some embodiments, the extracellular domain of the chimeric antigen receptor on CAR T cells is a polypeptide that specifically binds to one or more cell surface molecules (e.g., tumor antigens) that are preferentially or uniquely expressed on the extracellular surface of neoplastic cells, selected from the group consisting of GD2, BCMA, CD19, PSMA, CD33, CD38, CD70, GD2, IL3R□2, CD2, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP.

[0232] physical method In some embodiments, the supplementary agents are antineoplastic physical methods including, but not limited to, radiotherapy, cryotherapy, hyperthermia, surgery, laser ablation, and proton therapy.

[0233] formulation This disclosure further provides pharmaceutically acceptable formulations of the IL10Rb-binding molecules of this disclosure. Preferred formulations depend on the intended mode of administration and therapeutic application. The pharmaceutically acceptable forms of the IL10Rb-binding molecules described herein include inherently non-toxic and non-therapeutic physiologically acceptable carriers. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, e.g., human serum albumin, buffers, e.g., phosphates, glycine, sorbic acid, potassium sorbate, partially glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, and PEG. Carriers for topical or gel-based polypeptides include polysaccharides, such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, polymerizable amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0234] Pharmaceutical compositions may also contain pharmaceutically acceptable, non-toxic carriers, excipients, stabilizers, or diluents, which are defined as media commonly used to formulate pharmaceutical compositions for animal or human administration. Diluents are selected so as not to affect the biological activity of the combination. Acceptable carriers, excipients, or stabilizers are generally non-toxic to the recipient at the doses and concentrations used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, alkylparabens such as butyl or benzyl alcohol, methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; blood Proteins such as clear albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0235] Formulations used for in vivo administration are typically sterile. Sterilization of the compositions of this disclosure can be easily achieved by filtration through a sterile filtration membrane.

[0236] Typically, the compositions are prepared as injectable preparations, either as liquid solutions or suspensions; solid forms suitable for dissolving or suspending in a liquid medium before injection may also be prepared. Preparations may also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides, or copolymers for enhanced adjuvant effects, as discussed above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The agents of this disclosure may be administered in the form of depot injections or implant preparations, which may be formulated in a manner that allows for sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are generally formulated as sterile, substantially isotonic preparations in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0237] Vector delivery of polypeptide IL10Rb-binding molecules In embodiments where the IL10Rb-binding molecule is a polypeptide, such an IL10Rb-binding molecule may also be delivered to the subject via administration of a recombinant vector containing a nucleic acid sequence encoding a peptidyl IL10Rb-binding molecule functionally linked to an expression regulatory sequence in cells of the subject tissue.

[0238] The expression vector may be a viral vector or a nonviral vector. The term "nonviral vector" refers to an extrachromosomal circular DNA molecule that autonomously replicates, is distinct from the normal genome, is not essential for cell survival under nonselective conditions, and has the ability to result in the expression of a coding sequence in target cells. Plasmids are an example of a nonviral vector. To facilitate transfection of target cells, target cells may be directly exposed to a nonviral vector, which may be under conditions that promote the uptake of the nonviral vector. Examples of conditions that promote the uptake of foreign nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high levels of salt, and magnetic fields (electroporation).

[0239] In one embodiment, a nonviral vector may be provided in a nonviral delivery system. The nonviral delivery system is typically a complex for promoting transduction into target cells with a nucleic acid cargo, where the nucleic acid is complexed with an agent, such as cationic lipids (DOTAP, DOTMA), surfactants, biological agents (gelatin, chitosan), metals (gold, magnetite), and synthetic polymers (PLG, PEI, PAMAM). Numerous embodiments of nonviral delivery systems are well known in the art, including lipid vector systems (Lee et al. (1997) Crit Rev Ther Drug Carrier Syst. 14:173-206); polymer-coated liposomes (Marin et al., U.S. Patent No. 5,213,804, issued May 25, 1993; Woodle, et al., U.S. Patent No. 5,013,556, issued May 7, 1991); cationic liposomes (Epand et al., U.S. Patent No. 5,283,185, issued February 1, 1994; Jessee, JA, U.S. Patent No. 5,578,475, issued November 26, 1996; Rose et al., U.S. Patent No. 5,279,833, issued January 18, 1994; Gebeyehu et al., issued August 2, 1994) Including al., U.S. Patent No. 5,334,761.

[0240] In another embodiment, the expression vector may be a viral vector. As used herein, the term viral vector is used in its conventional sense and refers to any obligate intracellular parasite that has neither a protein synthesis mechanism nor an energy production mechanism, and generally refers to any enveloped or non-enveloped animal virus commonly used to deliver foreign transgenes to mammalian cells. Viral vectors may be replication competent (e.g., substantially wild-type), conditionally replicable (engineered by recombination to replicate under certain conditions), or replication-deficient (substantially lacking the ability to replicate in the absence of a cell line capable of compensating for the virus's missing function). Viral vectors may have certain modifications to make them "specifically replicable," i.e., preferentially replicate in a particular cell type or phenotypic cellular state, e.g., cancerous. Viral vector systems useful in the implementation of the IL10Rb-binding molecules of this disclosure include, for example, naturally occurring or recombinant viral vector systems. Examples of viruses useful in the implementation of the IL10Rb-binding molecules of this disclosure include recombinantly modified enveloped or non-enveloped DNA and RNA viruses. For example, viral vectors can be derived from the genomes of human or bovine siadenovirus, vaccinia virus, lentivirus, herpesvirus, adeno-associated virus, human immunodeficiency virus, Sindbisvirus, and retroviruses (including, but not limited to, Roussarcoma virus), and hepatitis B virus. Typically, the gene of interest is inserted into such a vector, which typically has an associated viral genome sequence, to enable the packaging of a gene construct, and subsequent infection of susceptible host cells results in the expression of the gene of interest (e.g., a targeted antigen).

[0241] The expression vector may encode one or more polypeptides in addition to the targeted antigen. When multiple polypeptides are expressed in the implementation of the IL10Rb binding molecule of this disclosure, each polypeptide may be functionally linked to an expression regulatory sequence (monocistronic), or the multiple polypeptides may be encoded by a polycistronic construct in which the multiple polypeptides are expressed under the control of a single expression regulatory sequence. In one embodiment, the expression vector encoding the targeted antigen may optionally further encode one or more immunological modulators. Examples of immunological modulators useful in the implementation of the IL10Rb binding molecule of this disclosure include, but are not limited to, cytokines. Examples of such cytokines include, but are not limited to, one or more of IL-1, IL-2, IL-3, IL-4, IL-12, interleukins, TNF-alpha, interferon-alpha, interferon-alpha-2b, interferon-beta, interferon-gamma, GM-CSF, MIP1-alpha, MIP1-beta, MIP3-alpha, TGF-beta, and other suitable cytokines having the ability to modulate an immune response. The expressed cytokines can be directed for intracellular expression or expressed in conjunction with signaling sequences for extracellular presentation or secretion.

[0242] The expression vector may optionally provide an additional expression cassette containing a nucleic acid sequence encoding a “rescue” gene. The “rescue gene” is a nucleic acid sequence whose expression makes the cell susceptible to catalytic killing or induces toxic conditions in the cell so that the cell is killed. The provision of a rescue gene enables selective cytotoxicity of the transduced cell. Thus, the rescue gene provides an additional safety precaution when the construct is incorporated into cells of mammalian target, preventing undesirable expansion of the transduced cell or the effects of the replication-competent vector system. In one embodiment, the rescue gene is a thymidine kinase (TK) gene (see, for example, Woo, et al., U.S. Patent No. 5,631,236, published May 20, 1997, and Freeman, et al., U.S. Patent No. 5,601,818, published February 11, 1997), and cells expressing the TK gene product become susceptible to selective killing by ganciclovir administration.

[0243] Dosage This disclosure further provides the administration of recombinant vectors or cells containing an effective therapeutic or prophylactic dose of an IL10Rb-binding molecule or a nucleic acid sequence encoding a polypeptide IL10Rb-binding molecule to subjects who are suffering from or at risk of developing a disease, disorder, or condition. The dosage of a pharmaceutical composition containing an IL10Rb-binding molecule, vector, or cells depends on factors including the route of administration, the disease to be treated, and the physical characteristics of the subject, such as age, weight, and overall health status. Typically, the amount of IL10Rb-binding molecule contained in a single dose may be an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity. The pharmaceutical compositions disclosed herein are available in concentrations of 0.01 to 500 mg / kg (e.g., 0.01 to 450 mg, 0.01 to 400 mg, 0.01 to 350 mg, 0.01 to 300 mg, 0.01 to 250 mg, 0.01 to 200 mg, 0.01 to 150 mg, 0.01 to 100 mg, 0.01 to 500 mg). mg / kg, 1~500mg / kg, 5~500mg / kg, 10~500mg / kg, 50~500mg / kg, 100~500mg / kg, 150~500mg / kg , 200~500mg / kg, 250~500mg / kg, 300~500mg / kg, 350~500mg / kg, 400~500mg / kg, or 450~500mg In a more specific embodiment, the dosage of the IL10Rb binding molecule described herein may include doses ranging from about 1 to about 100 mg / kg (e.g., about 1 to about 90 mg / kg, about 1 to about 80 mg / kg, about 1 to about 70 mg / kg, about 1 to about 60 mg / kg, about 1 to about 50 mg / kg, about 1 to about 40 mg / kg, about 1 to about 30 mg / kg, about 1 to about 20 mg / kg, about 1 to about 10 mg / kg, about 10 to about 100 mg / kg, about 20 to about 100 mg / kg, about 30 to about 100 mg / kg, about 40 to about 100 mg / kg, about 50 to about 100 mg / kg, about 60 to about 100 mg / kg, about 70 to about 100 mg / kg, about 80 to about 100 mg / kg, or about 90 to about 100 mg / kg).In some embodiments, the pharmaceutical compositions of this disclosure may include dosages of binding proteins described herein ranging from 0.01 to 20 mg / kg (e.g., 0.01 to 15 mg / kg, 0.01 to 10 mg / kg, 0.01 to 8 mg / kg, 0.01 to 6 mg / kg, 0.01 to 4 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg, 0.01 to 0.05 mg / kg, 0.05 to 20 mg / kg, 0.1 to 20 mg / kg, 1 to 20 mg / kg, 2 to 20 mg / kg, 4 to 20 mg / kg, 6 to 20 mg / kg, 8 to 20 mg / kg, 10 to 20 mg / kg, 15 to 20 mg / kg). The dosage may be adapted by a physician according to conventional factors, such as the severity of the disease and different parameters of the subject.

[0244] Pharmaceutical compositions containing the IL10Rb binding molecule described herein may be administered, for example, once or multiple times (e.g., 1 to 10 times or more) daily, weekly, monthly, twice a year, annually, or as medically required to a subject requiring it. Dosages may be provided in a single or multiple dosing regimen. The timing between administrations may decrease as the medical condition improves or increase as the patient's health deteriorates. The course of therapy may be a single dose or multiple doses over a period of time. In some embodiments, a single dose is used. In some embodiments, two or more divided doses are used, administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. Each dose administered in such divided dosing protocols may be the same or different in each administration. Multi-day medication protocols over a period of time may be provided by those skilled in the art who monitor the administration, taking into account the adverse effects of the treatments discussed above and the subject's response to the treatment, including their modification.

[0245] For prophylactic applications, a pharmaceutical composition or medicine is administered in a sufficient amount to patients who are susceptible to the disease or otherwise at risk thereof, to eliminate or reduce the risk of the disease, decrease its severity, or delay its onset, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear between the onset of the disease.

[0246] In some embodiments, the condition to be treated is a chronic condition (e.g., a chronic infection, i.e., an infection that is not cleared by the host immune system within a period of one, two, or more weeks). In some cases, the chronic condition involves the incorporation of pathogenic genetic elements into the host genome, such as retroviruses, lentiviruses, or hepatitis B viruses. In other cases, chronic infections, such as those caused by certain intracellular bacteria or protozoan pathogens, result from the presence of pathogenic cells within host cells. Additionally, in some embodiments, the infection is in its incubation period, such as with herpesviruses or human papillomaviruses. In such cases, the course of therapy may involve the administration of IL10Rb-binding molecules over a long period, including continued administration in the substantial absence of symptoms of the chronic condition to prevent recurrence of the chronic condition or its symptoms.

[0247] In prophylactic applications, relatively low doses may be administered over a long period of time at relatively low intervals. Some patients may continue treatment for the rest of their lives. In other therapeutic applications, relatively high doses at relatively short intervals may be required until disease progression is reduced or terminated, preferably until the patient experiences partial or complete remission of the disease symptoms. Thereafter, the patient may be administered a prophylactic regimen.

[0248] Route of administration The administration of the IL10Rb-binding molecules described herein may be achieved by any of the various methods known in the art, including, but not limited to, topical application, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodular injection, percutaneous, transmucosal, iontophoretic delivery, intralymphatic injection (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), respiratory inhaler including nebulizer, intraocular injection, intraperitoneal injection, intrafocal injection, intraovarian injection, intracerebral infusion or injection, and intraventricular injection (ICVI). Administration to the subject may be achieved by intravenous infusion as a bolus or by continuous infusion over a period of time. Examples of parenteral administration routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. IL10Rb-binding molecules may be administered once, continuously, for example by a continuous pump, or at regular intervals over a period of time that may be one week, two weeks, one month, two months, three months, or longer (e.g., daily, every two weeks, monthly). Desired time intervals for multiple doses of IL10Rb-binding molecules can be determined by those skilled in the art.

[0249] As described herein, the compositions of this disclosure may be used in combination with one or more additional therapeutically effective agents. Where used herein, the term “in combination with” in reference to the administration of multiple agents to a subject means the administration of the first agent with at least one additional (i.e., second, third, fourth, fifth, etc.) supplementary agent to the subject. For the purposes of this disclosure, one agent (e.g., an IL10Rb binding molecule) is considered to be administered in combination with a supplementary agent if the biological effect resulting from the administration of the first agent is sustained in the subject at the time of administration of the supplementary agent, such that the therapeutic effects of the first and second agents overlap. The administration of the first agent may provide a therapeutic effect over a long period of time, and the administration of the supplementary agent may provide its therapeutic effect while the therapeutic effect of the first agent is still ongoing, and as a result, the supplementary agent is considered to be administered in combination with the first agent even if the first agent is administered at a point in time significantly far removed from the time of administration of the supplementary agent (e.g., days or weeks). In one embodiment, one agent is considered to be administered in combination with a supplementary agent when the first and second agents are administered simultaneously (within 30 minutes of each other), concurrently, or sequentially. In some embodiments, the first agent is considered to be administered “contemporarily” with the supplementary agent when the first and supplementary agents are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other. The term “in combination” is also understood to apply to situations in which the first agent and the supplementary agent are co-formulated in a single pharmaceutically acceptable formulation and the co-formulation is administered to the subject. In certain embodiments, the first agent and the supplementary agent are administered or applied sequentially, for example, one agent is administered before one or more other agents. In other embodiments, the first agent and the supplementary agent are administered simultaneously, for example, two or more agents are administered at the same or approximately the same time; two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation).Whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0250] kit This disclosure also envisions a kit comprising a pharmaceutical composition of an IL10Rb-binding molecule. In some embodiments, the kit further comprises a supplementary pharmaceutical composition comprising the supplementary agents discussed above for use in combination therapy with the IL10Rb-binding molecule. The kit generally takes the form of a physical structure containing the various components described below, which may be used, for example, in the implementation of the methods described above. The kit may include the IL10Rb-binding molecule in the form of a pharmaceutical composition suitable for administration to a subject that is prepared for use, or in a form that requires pre-administration preparation, e.g., thawing, reconstitution, or dilution. If the IL10Rb-binding molecule is in a form that requires reconstitution by the user, the kit may also include a sterile container providing a reconstitution medium containing a buffer and pharmaceutically acceptable excipients, etc. The kits of this disclosure may be designed for conditions necessary to properly maintain the components contained in the kit (e.g., freezing or refrigeration). The kit may further comprise a label or accompanying document containing identifying information of the components contained in the kit, and instructions for use. Each component of the kit may be sealed in an individual container, or all the various containers may be in a single packaging container. The label or accompanying information may contain manufacturer information such as lot number and expiration date. The label or accompanying information may be, for example, integrated into the surface of the physical structure containing the components, placed separately within the physical structure, or affixed to the components of the kit (e.g., ampoules, syringes, or vials). The label or accompanying information may be provided in physical form or on a computer-readable medium. In some embodiments, the actual instructions are not present in the kit, and the kit provides means for obtaining the instructions from a remote source, for example, via an internet site, including obtaining the instructions by secure access through the provision of a password (or a scannable code such as a barcode or QR code on the surface of the container of the IL10Rb binding molecule or the kit containing it), in accordance with government regulations (e.g., HIPAA). [Examples]

[0251] The following examples are provided to fully disclose and illustrate to those skilled in the art how to prepare and use the IL10Rb-binding molecule, and are not intended to limit the scope of what the inventors consider to be an IL10Rb-binding molecule, nor are they intended to indicate that the following experiments have been performed or are all possible experiments. Illustrative descriptions written in the present tense are not necessarily performed, but should be understood as being possible to obtain the data, etc., described herein. Efforts have been made to ensure accuracy regarding the numerical values ​​used (e.g., quantities, temperatures, etc.), but some experimental error and deviation are to be expected. Variations of the detailed procedures used may become apparent to those skilled in the art, and it is expected that such variations can be used as appropriate. Thus, the IL10Rb-binding molecule may be prepared in ways other than those described herein, and the present invention is intended to include all modifications and equivalents of the subject matter described in the claims added to the end of this specification, as permitted by applicable law.

[0252] Unless otherwise specified, parts are measured by weight, molecular weight is the weight-average molecular weight, temperature is in degrees Celsius (°C), and pressure is atmospheric pressure or close to atmospheric pressure. Standard abbreviations are used, including: bp = base pair (s); kb = kilobase (s); pl = picoliter; s or sec = second; min = minute; h or hr = hour; aa = amino acid (s); kb = kilobase (s); nt = nucleotide (s); pg = picogram; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar concentration; mM = millimolar concentration; M = molar concentration; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = once daily; BID =Twice a day; QW =Once a week; QM =Once a month; HPLC = High-performance liquid chromatography; BW = Body weight; U = Units; ns = Not statistically significant; PBS = Phosphate-buffered saline; PCR = Polymerase chain reaction; NHS = N-hydroxysuccinimide; HSA = Human serum albumin; MSA = Mouse serum albumin; DMEM = Dulbecco's modified Eagle medium; GC = Genome copy; EDTA = Ethylenediaminetetraacetic acid; PBMC = Primary peripheral blood mononuclear cells; FBS = Fetal bovine serum; FCS = Fetal calf serum; HEPES = 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; LPS = Lipopolysaccharide; ATCC = American Type Culture Collection.

[0253] Example 1. Immunotherapy protocol for the generation of hIL10Rb and mIL10Rb VHH The isolation of anti-hIL10Rb VHH was initiated by immunization of camels with the extracellular domain of 201 amino acids of hIL10Rb, amino acids 20-220 of the precursor, and amino acids 1-201 of the mature protein (UNIPROT Reference No. Q08334). The isolation of anti-mIL10Rb VHH was initiated by immunization of camels with the extracellular domain of 201 amino acids of mIL10Rb, amino acids 20-220 of the precursor, and amino acids 1-201 of the mature protein (UNIPROT Reference No. Q61190). For each antigen, VHH was identified and isolated using the following methodology.

[0254] The synthetic DNA sequence encoding the antigen was inserted into the pFUSE_hIgG1_Fc2 vector (Generay Biotechnology) and transfected into HEK293F mammalian cell host cells for expression. The antigen was expressed as an Fc fusion protein purified using protein A chromatography. The antigen was diluted with 1×PBS (approximately 1 mg of antigen in total). Quality was assessed by SDS-PAGE to ensure sufficient purity for immunization (>80%). Camels were acclimatized in the facility for at least 7 days prior to immunization. Immunization with the antigen was performed using weekly antigen administration for 7 weeks. For the initial immunization, the immunogen was prepared as follows: 10 mL of complete Freund's adjuvant (CFA) was added to a mortar, and then 10 mL of antigen dissolved in 1×PBS was slowly added to the mortar while grinding with a pestle until the antigen emulsified, became milky white, and was difficult to disperse. Subsequently, the immunogen was prepared as described above, except that incomplete Freund's adjuvant (IFA) was used instead of CFA for six immunizations (weeks 2-7) in the immunization protocol. Approximately 2 ml of emulsified antigen was subcutaneously injected into at least six sites on the camels, with a total of approximately 10 mL of emulsion per camel. To avoid leakage of the emulsion after each injection, the needle was kept in the subcutaneous space for approximately 10-15 seconds.

[0255] Example 2. Phage library construction In the immunization protocol, blood samples were collected from camels three days after the final injection. RNA was extracted from the blood and transcribed into cDNA. From a desirable approximately 700 bp fragment encoding the VHH-Hinge-CH2-CH3 species, an approximately 900 bp reverse transcription sequence encoding the VH-CH1-Hinge-CH2-CH3 construct was isolated. The purified approximately 700 bp fragment was amplified by nested PCR. The amplified sequence was digested with Pst1 and Not1. An approximately 400 bp Pst1 / Not1 digested fragment was inserted into a Pst1 / Not1 digested pMECS phagemide vector so that the VHH coding sequence was in-frame with the DNA sequence encoding the HA / His sequence. The sequence produced by PCR and the pMECS phagemide vector were digested with PstI and NotI, and then ligated to pMECS / Nb recombination. After ligation, the product was introduced into E. coli (E. coli) TG1 cells by electroporation transformation. The transformants were concentrated in growth medium and then transferred to 2YT + 2% glucose agar plates.

[0256] Example 3: Isolation of antigen-specific VHH Biopanning of phage libraries was performed to identify VHH that binds to IL10Rb. 96-well plates were coated with IL10Rb, and phage libraries were incubated in each well to allow phage-expressed IL10Rb-reactive VHH to bind to the IL10Rb on the plate. Non-specifically bound phages were washed away, and specifically bound phages were isolated. After selection, the enriched phage libraries expressing IL10Rb-reactive VHH were amplified in TG1 cells. The biopanning process described above was repeated 2-3 times to enrich the library for IL10Rb-reactive VHH.

[0257] Example 4: Identification of antibodies that specifically bind to IL10Rb: After the biopanning in Example 3 was completed, three 96-well plates of individual phage clones were isolated by periplasmic extract ELISA (PE-ELISA) on IL10Rb-coated plates to identify positive VHH conjugates that selectively bound to IL10Rb. The 96-well plates were coated with IL10Rb and PBS under the same conditions. The wells were then blocked at 37°C for 1 hour. Next, 100 μl of extracted antibody was added to each well and incubated for 1 hour. Subsequently, 100 μl of HRP-conjugated anti-tag polyclonal antibody was added to each well and incubated at 37°C for 1 hour. The plates were stained with TMB substrate. The reaction was stopped by adding H2SO4. Absorbance at 450 nm was read using a microtiter plate reader. Antibodies whose absorbance in the antigen-coated well was at least 3 times that of the PBS-coated control were VHHs that specifically bound to IL10Rb. Positive clones were sequenced, and their sequences were analyzed to identify unique chronotypes.

[0258] Example 5. Evaluation of coupling affinity via surface plasmon resonance To evaluate the binding via SPR as follows, one representative example was selected from each clonotype of the hIL10Rb VHHs prepared according to Examples 1 to 3. The binding affinity of the IL10Rb-binding molecules to hIL10Rb corresponding to SEQ ID NO: 109, 120, 111, 123, 124, 134, and 135 was evaluated using surface plasmon resonance (SPR) generally according to the following procedure. All experiments were performed on a Biacore T200 instrument equipped with a Protein A-derivatized sensor chip (Cytiva) in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20), and 3 mM EDTA (HBS-EP+ buffer). The mono-Fc VHH ligand was flowed at 5 μl / min for a variable time of 18 - 300 s to reach the capture load listed in the following table. After ligand capture, a two-fold dilution series of the extracellular domain of the IL10Rb receptor modified to incorporate a C-terminal poly-His sequence, typically containing at least 5 concentrations from 1 μM to 1 nM, was injected in high performance mode or single cycle kinetics mode. Surface regeneration was performed by flowing 10 mM glycine-HCl, pH 1.5 (60 s, 50 μL / min). The sensogram minus buffer was processed with Biacore T200 Evaluation Software and globally fit using a 1:1 Langmuir binding model (setting bulk shift to 0) to extract the rate constants and affinity constants (k a , k d , K D ). R MAX <100 RU indicates a surface density compatible with the reaction rate analysis. R max The calculated values were generated using the formula: Rmax = load (RU) x valence of the ligand x (molecular weight of the analyte / molecular weight of the ligand). The surface activity was defined as the ratio of the experimental Rmax value to the calculated value. The results of these binding affinity experiments are shown in Table 6 above.

[0259] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art in consideration of the examples and embodiments described herein and will be understood to be included in the spirit and scope of this application and the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0260] Sequence information SEQUENCE LISTING <110> SYNTHEKINE, INC. <120> IL10RB BINDING MOLECULES AND METHODS OF USE <150> US 63 / 135,884 <151> 2021-01-11 <150> US 63 / 078,745 <151> 2020-09-15 <150> US 63 / 061,562 <151> 2020-08-05 <160> 175 <170> PatentIn version 3.5 <210> 1 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Poly <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Gly 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ala Ala Ile Asn Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 2 Tyr Thr Tyr Ser Ser Gly Cys Met Gly 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Ala Ile Asn Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu Phe 1 5 10 15 Gly Tyr <210> 5 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Lys Arg Thr Met Val Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Glu Pro Tyr Cys Ser Gly Gly Tyr Lys Arg Thr Met Val Ala Glu Phe 1 5 10 15 Gly Tyr <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Tyr Asn Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Asp Ser Asp Gly Met Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Ala Asp Cys Thr Ile Ala Ala Met Thr Thr Asn Pro Leu Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Tyr Thr Tyr Asn Ser Tyr Cys Met Gly 1 5 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Thr Ile Asp Ser Asp Gly Met Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Asp Ala Asp Cys Thr Ile Ala Ala Met Thr Thr Asn Pro 1 5 10 <210> 13 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ile Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Leu Tyr Ser Ile Asp 20 25 30 Tyr Met Ala Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Pro Val 35 40 45 Ala Val Ile Tyr Thr Ala Ser Gly Ala Thr Phe Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Met Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Lys Thr Asp Ser Tyr Leu Phe Asp Ala Gln Ser Phe 100 105 110 Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Tyr Leu Tyr Ser Ile Asp Tyr Met Ala 1 5 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Val Ile Tyr Thr Ala Ser Gly Ala Thr Phe Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Val Arg Lys Thr Asp Ser Tyr Leu Phe Asp Ala Gln Ser Phe Thr Tyr 1 5 10 15 <210> 17 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys Tyr 1 5 10 15 <210> 21 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ile Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Asp Leu Tyr Ser Thr Asn 20 25 30 Tyr Val Ala Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ala Val Ile Tyr Thr Ala Ser Gly Ala Thr Leu Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Met Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Lys Thr Gly His Tyr Leu Phe Asp Ala Gln Ser Phe 100 105 110 Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Asp Leu Tyr Ser Thr Asn Tyr Val Ala 1 5 <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Val Ile Tyr Thr Ala Ser Gly Ala Thr Leu Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Val Arg Lys Thr Gly His Tyr Leu Phe Asp Ala Gln Ser Phe Thr Tyr 1 5 10 15 <210> 25 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Gly 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Asn Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Tyr Thr Tyr Ser Ser Gly Cys Met Gly 1 5 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Thr Ile Asn Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 28 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu Phe 1 5 10 15 Gly Tyr <210> 29 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ala Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ala Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Trp Cys Thr Gly Gly Tyr Ser Arg Leu Thr Pro Ala Glu 100 105 110 Tyr Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Tyr Ser Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Ala Ile Ala Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 32 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Glu Pro Trp Cys Thr Gly Gly Tyr Ser Arg Leu Thr Pro Ala Glu Tyr 1 5 10 15 Gly Tyr <210> 33 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Gly 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Asn Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Tyr Thr Tyr Ser Ser Gly Cys Met Gly 1 5 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Thr Ile Asn Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 36 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu Phe 1 5 10 15 Gly Tyr <210> 37 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys Tyr 1 5 10 15 <210> 41 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Lys Arg Thr Met Val Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 44 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Glu Pro Tyr Cys Ser Gly Gly Tyr Lys Arg Thr Met Val Ala Glu Phe 1 5 10 15 Gly Tyr <210> 45 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 47 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys Tyr 1 5 10 15 <210> 49 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Asn 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Asn Tyr 1 5 10 15 <210> 53 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Gly 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Asn Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Tyr Thr Tyr Ser Ser Gly Cys Met Gly 1 5 <210> 55 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Thr Ile Asn Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 56 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Glu Pro Tyr Cys Ser Gly Gly Tyr Pro Arg Trp Ser Val Ala Glu Phe 1 5 10 15 Gly Tyr <210> 57 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Arg Tyr Thr Ala Ser Val Asn 20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Phe Thr Gly Ala Gly Thr Thr Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Val Asp Phe Arg Gly Gly Leu Leu Tyr Arg Pro Ala Tyr Glu Tyr 100 105 110 Thr Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Tyr Thr Ala Ser Val Asn Tyr Met Gly 1 5 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Thr Ile Phe Thr Gly Ala Gly Thr Thr Tyr Tyr Ala Asn Ser Val Lys 1 5 10 15 Gly <210> 60 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Asp Phe Arg Gly Gly Leu Leu Tyr Arg Pro Ala Tyr Glu Tyr Thr Tyr 1 5 10 15 <210> 61 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 61 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr His Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Val Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Phe Ala Asp Cys Ser Ser Asn Tyr Phe Leu Pro Pro Gly Ala 100 105 110 Val Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Tyr Thr His Ser Ser Tyr Cys Met Gly 1 5 <210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Ala Ile Asp Val Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 64 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Glu Phe Ala Asp Cys Ser Ser Asn Tyr Phe Leu Pro Pro Gly Ala Val 1 5 10 15 Arg Tyr <210> 65 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 65 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Arg Tyr Thr Ala Ser Val Asn 20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Phe Thr Gly Ala Gly Thr Thr Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Val Asp Phe Arg Gly Gly Leu Leu Tyr Arg Pro Ala Tyr Glu Tyr 100 105 110 Thr Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Tyr Thr Ala Ser Val Asn Tyr Met Gly 1 5 <210> 67 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Thr Ile Phe Thr Gly Ala Gly Thr Thr Tyr Tyr Ala Asn Ser Val Lys 1 5 10 15 Gly <210> 68 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Asp Phe Arg Gly Gly Leu Leu Tyr Arg Pro Ala Tyr Glu Tyr Thr Tyr 1 5 10 15 <210> 69 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 69 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Phe Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Glu 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr His Arg Lys Glu Met Ala Glu 100 105 110 Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Asp Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 71 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Phe Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Glu Gly 1 5 10 15 <210> 72 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 72 Glu Pro Tyr Cys Ser Gly Gly Tyr His Arg Lys Glu Met Ala Glu Phe 1 5 10 15 Gly Tyr <210> 73 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 73 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys 100 105 110 Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 74 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 76 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 76 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys Tyr 1 5 10 15 <210> 77 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 77 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 78 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 79 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys Tyr 1 5 10 15 <210> 81 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 81 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Tyr Thr Ala Ser Asn Asn 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Phe Thr Gly Ala Gly Thr Ser Tyr Tyr Asp Ser Ser Val 50 55 60 Gly Arg Leu Phe Ile Ser Ser Gln Asp Ala Ala Ser Thr Leu Asp Gln 65 70 75 80 Leu Leu Met Ser Leu Leu Pro Asp Asp Thr Ala Val Met Tyr Cys Gly 85 90 95 Ala Glu Asp Asp Cys Thr Leu Leu Leu Met Thr Pro Asn Pro Asp Asp 100 105 110 Gln Trp Ser Arg Leu Ser Val Ser Ser 115 120 <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 82 Tyr Thr Ala Ser Asn Asn Cys Met Gly 1 5 <210> 83 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Val Ile Phe Thr Gly Ala Gly Thr Ser Tyr Tyr Asp Ser Ser Val Gly 1 5 10 15 <210> 84 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Glu Asp Asp Cys Thr Leu Leu Leu Met Thr Pro Asn Pro Asp Asp Gln 1 5 10 15 <210> 85 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 85 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asp Ser Arg Tyr 20 25 30 Cys Met Gly Trp Phe Arg Lys Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Tyr Thr Asp Ser Arg Tyr Cys Met Gly 1 5 <210> 87 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 His Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 88 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Lys Tyr 1 5 10 15 <210> 89 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 89 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Lys Ile Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Val Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Tyr Cys Ser Gly Gly Tyr Lys Arg Thr Met Val Ala Glu 100 105 110 Phe Gly Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 91 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Ala Ile Asp Ser Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 92 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Glu Pro Tyr Cys Ser Gly Gly Tyr Lys Arg Thr Met Val Ala Glu Phe 1 5 10 15 Gly Phe <210> 93 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 93 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Asn 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 94 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 95 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 95 His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 96 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 96 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Asn Tyr 1 5 10 15 <210> 97 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 97 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile 35 40 45 Ala His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Asn 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 98 Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 99 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 99 His Ile Asp Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 100 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 100 Asp Pro Ile Pro Gly Pro Gly Tyr Cys Asp Gly Gly Pro Asn Asn Tyr 1 5 10 15 <210> 101 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 101 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Asp Ser Asp Gly Met Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Pro Leu Tyr Asp Cys Asp Ser Gly Ala Val Gly Arg Asn Pro Pro 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 102 Tyr Ser Tyr Ser Ser Tyr Cys Met Gly 1 5 <210> 103 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Thr Ile Asp Ser Asp Gly Met Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 104 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 104 Pro Leu Tyr Asp Cys Asp Ser Gly Ala Val Gly Arg Asn Pro Pro Tyr 1 5 10 15 <210> 105 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 105 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Leu Arg Gly 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Met Asp Val Val Gly Asp Arg Arg Ser Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Ser Val Tyr 65 70 75 80 Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Ala Gly Pro Asn Cys Val Gly Trp Arg Ser Gly Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 106 Tyr Thr Tyr Leu Arg Gly Cys Met Gly 1 5 <210> 107 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 107 Val Met Asp Val Val Gly Asp Arg Arg Ser Tyr Ile Asp Ser Val Lys 1 5 10 15 Gly <210> 108 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Gly Pro Asn Cys Val Gly Trp Arg Ser Gly Leu Asp Tyr 1 5 10 <210> 109 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 109 caggtgcagc ttcaggaatc aggcggaggc agcgtgcagg cagggggtag cctgcgtctg 60 tcttgcgcag ccagcgggta cacctacagc tctggctgta tgggctggtt tcgccaagcc 120 ccaggaaaag aacgggaagc cgtggcggct atcaatagcg acggctccac ctcctatgct 180 gactccgtca aaggacgctt caccattagt aaagataacg ccaagaacac cttgtacctt 240 cagatgaact ccttgaaacc ggaggacacc gcaatgtatt actgtgcggc tgagccctac 300 tgctcaggag gctacccacg gtggtcagtg gccgagtttg gttattgggg ccagggcacc 360 caagtgactg tgtcctcc 378 <210> 110 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 110 caggtgcaac tccaggagtc agggggaggt tccgtgcagg ctggcggttc tctcaggttg 60 tcttgcgcgg ccagcggcta tacgtacagt agctactgca tgggctggtt ccggcaagcc 120 cccggcaagg agcgcgaagg cgtggctgcc attgattccg atggatctac taggtatgct 180 gatagtgtaa agggccgctt cacaatctcc aaggacaatg ccaagaacac actgtatttg 240 caaatgaact ccctcaagcc cgaggatacc gctatgtact attgcgctgc cgaaccatac 300 tgttccggtg gctataagcg cactatggtg gccgagttcg gatactgggg tcaaggcaca 360 caggtcacag tgtcctct 378 <210> 111 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 111 caggtgcagt tgcaggagtc cgggggcggt agcgttcagg ctggagggtc cctgcgtctg 60 agttgtgcgg catctcggta tacttataac agttactgta tgggttggtt ccgccaggca 120 cctggaaagg agcgggaggg ggtggcgact attgatagcg acggaatgac cagatatgcc 180 gactctgtga agggaagatt tactatctca aaagataatg ccaagaacac actctatttg 240 cagatgaaca gcctcaagcc agaggatacc gctatgtatt actgtgctgc cgacgctgat 300 tgcaccatcg ctgccatgac gaccaacccc ttgggccagg gaacccaagt aaccgtctct 360 agc 363 <210> 112 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 112 caggtccagc tccaggaatc tggtggcggg tctatccagg cgggtggcag cctgcggctg 60 agttgcgccg cttcccgcta cctgtatagt attgattata tggcctggtt caggcagtca 120 ccgggcaaag agcgcgaacc cgtcgctgtg atttacacag cctctggtgc caccttctat 180 cccgatagtg tgaagggccg gttcactatc tctcaagaca acgcgaagat gactgtctat 240 cttcagatga actctctgaa gtccgaggac actgccatgt attactgtgc cgctgtgcgc 300 aagacggact cttatctgtt cgatgcccag agtttcactt actggggtca gggtactcag 360 gtgaccgtat cctcc 375 <210> 113 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 113 caggtgcagc tccaggagtc tggtggcggg ctggttcagc ctgggggttc actccggttg 60 tcctgcgctg cgtctggtta tacctactcc agctactgta tgggttggtt ccgccaggca 120 ccggggaagg agagggaggg cgtggctcac attgattctg atggctctac gacctacgct 180 gatagcgtta aggggcgctt cactatctcc aaggataacg ccaagaacac cctgtatctg 240 caaatgaaca gcctgaagcc agaagacact gccatgtact attgcgctgc cgatcctatt 300 cccggtcctg gctattgtga cggcggtcct aacaagtact ggggccaagg cacacaggtg 360 actgtcagtt cc 372 <210> 114 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 114 caggttcaac tccaggaatc cggcggtgga agcattcagg cgggcggttc tttgactctg 60 agctgtgcgg catctcggga cctttacagc actaactatg ttgcctggtt ccggcagtcc 120 cccggcaagg aacgcgaagc tgtggccgtg atttatacag ccagcggcgc aaccctgtat 180 agcgattcag tcaaaggccg gttcaccatc tcccaggaca acgcgaagat gaccgtgtac 240 ctgcaaatga acagcctgaa gtctgaggac actgccatgt attactgcgc agctgtgaga 300 aagaccggac attacctctt cgacgcccaa tctttcacct actggggcca gggaacccag 360 gtcaccgtct cctct 375 <210> 115 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 115 caggtgcaac tccaggagtc aggcggtggg tccgtccagg ccggtggctc cctgaggctg 60 agttgcgccg cttccggcta tacttactcc agcggttgca tggggtggtt ccgccaagcc 120 cccggtaaag aacgcgaggg agtggctaca attaactccg atggaagcac taactacgcc 180 gactctgtga agggacgctt caccattagc aaagacaatg ctaagaacac cctttacctt 240 caaatgaaca gcctgaagcc tgaggatacc gctatgtatt actgtgccgc agaaccgtat 300 tgtagcggtg gctaccctcg ctggtccgtc gccgagttcg gttattgggg ccaggggacc 360 caagtgactg tttctagc 378 <210> 116 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 116 caggtgcaac ttcaggagag cggcgggggc tctgtgcaag ctggtggctc cctgcggctc 60 agctgtgctg cctctgggta ttcttacagt agctactgta tgggctggtt cagacaggca 120 ccaggcaagg agcgcgaggg tgtggcggcc atcgcttccg acgggagtac cagctacgcc 180 gacagcgtta aaggtaggtt tgccatctct aaggataatg cgaagaatac actctacctt 240 cagatggcta gtctgaagcc agaggatacc gccatgtatt actgcgcggc agagccctgg 300 tgcacgggag ggtattcacg cctgaccccg gctgagtatg gatactgggg gcagggcacc 360 caggtgaccg ttagctcc 378 <210> 117 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 117 caggtccagt tgcaggaaag cggagggggc ctggtgcagc caggaggttc tctgagactg 60 agctgtgccg cttctggtta cacatattct agcgggtgca tgggctggtt ccgccaggct 120 cccggcaagg aacgtgaggg tgtggcaact atcaattccg acggctctac aaactacgca 180 gattctgtta aaggccgctt cacaatctct aaggacaacg ccaaaaacac tctgtacttg 240 cagatgaata gcctgaagcc tgaagacact gccatgtact attgcgcagc tgagccctac 300 tgttctggag gctacccccg ctggtctgtg gccgagttcg gttactgggg acaaggaacc 360 caggtcacag tgtccagt 378 <210> 118 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 118 caggttcagc tccaggagtc aggcgggggt cttgtccagc ctggtggctc cctgcgcctg 60 tcctgtgctg cctccggtta cacctactcc agctattgca tgggatggtt cagacaagcg 120 ccaggcaagg aacgtgaggg ggtcgcccac attgactccg acggttccac tacctacgcc 180 gacagcgtca aaggccgctt cgcgatttct aaggataacg ctaagaatac tctgtacttg 240 cagatgaact cactgaagcc agaggacacg gccatgtatt actgcgcagc cgatccgatc 300 cccggccccg gctattgtga cggtggcccg aacaagtact ggggacaggg cacccaagtg 360 acggtgtcct ct 372 <210> 119 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 119 caggtacagt tgcaggagag cggaggcggt tccgtgcagg caggtggctc tcttagactg 60 tcctgcgccg cgagcgggta cacctacagt agctattgta tgggctggtt ccgccaggct 120 cctggtaagg gtcgcgaggg cgtcgctgcc atcgactccg atggctctac tcgctacgca 180 gattctgtca aggggcgctt cacaatttcc aaggacaacg ccaagaacac gctttacttg 240 cagatgaact cactgaagcc ggaggacacc gctatgtatt actgcgctgc cgagccctac 300 tgttctgggg gctacaagcg cactatggtg gccgagttcg gatattgggg ccagggtaca 360 caggtgaccg tcagttct 378 <210> 120 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 120 caggtgcagt tgcaggagtc tggcggtggc tctgtgcagg ctgggggctc tctgcgcctg 60 agttgcgctg ccagcggtta cacctactcc agctattgta tgggatggtt ccgccaggct 120 ccggggaagg agagggaggg cgtggcccat atcgactctg atggctccac atcctacgcc 180 gacagcgtga agggacgttt caccattagc aaggacaatg cgaagaatac cctctacttg 240 cagatgaact ccctgaagcc ggaggatact gccatgtatt actgcgccgc tgatcccatc 300 ccagggcctg ggtactgtga cggaggcccg aacaagtatt ggggacaagg aacgcaggtc 360 acagtgtcat ct 372 <210> 121 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 121 caggtacaac tccaggagag tggtggaggc tccgttcaag ccgggggctc cctgcggctg 60 tcctgtgcgg ccagcggtta cacctattca tcttactgta tgggctggtt ccggcaggcc 120 cctggtaagg aaagagaggg tgtcgctcac attgattccg acggtagtac ctcttacgca 180 gactctgtca agggcaggtt caccatctct aaggacaatg ccaagaacac cttgtacctc 240 cagatgaact ctctgaagcc cgaggacact gcaatgtact attgtgcggc tgaccctatt 300 cccggccctg gatattgcga cggcggacct aacaattact ggggacaggg cacccaggtc 360 accgtcagct cc 372 <210> 122 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 122 caggttcagc tccaagaatc cggcgggggc tctgtgcagg cgggcggaag tctgcgtctg 60 tcatgcgctg ccagcgggta cacttactct tccggttgta tgggctggtt taggcaggct 120 ccgggaaagg aaagggaggg cgtcgcaact atcaacagcg acggctctac gaactacgct 180 gactctgtga aaggccgctt caccatcagc aaagacaacg ccaaaaatac actgtatctc 240 cagatgaata gcttgaaacc cgaggacacc ggaatgtatt actgcgcggc agagccatac 300 tgttcaggcg gttacccaag atggtccgtg gctgagttcg gttattgggg gcagggcact 360 caggttactg tgtcttcc 378 <210> 123 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 123 caggtgcagc tccaggaatc cgggggcggt tctgtgcagg ctggtggctc tctgcgcctg 60 tcttgcactg tttccaggta cactgcctct gtaaactata tgggctggtt tagacaagct 120 ccgggcaagg aacgcgaagg cgtcgctacc atctttacag gtgcaggtac gacctattac 180 gccaatagcg ttaaagggag gttcaccatc tccagggaca atgccaaaaa cacagcctat 240 ctccagatga actccctcaa acctgaagac acagccatct actattgcgc ggttgacttc 300 cgtggtggcc tgctctatag accggcgtat gagtacacct accgtggaca aggcacccaa 360 gtcacagtga gcagc 375 <210> 124 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 124 caggtgcagc tccaagagtc cggcggaggg agtgtagagg ctggcgggtc cctgcgcctt 60 agctgcgcgg ccagcggcta tacacacagt tcttattgta tgggttggtt ccgccaagct 120 ccgggaaagg agcgtgaggg cgtggctgcc atcgacgtgg atggctccac aacctacgcc 180 gacagcgtga agggcaggtt tacgatctct aaggataacg ctaagaatac tctctatttg 240 cagatgaact ccctcaaacc cgaggataca ggaatgtact attgcgctgc cgagttcgcc 300 gactgctcaa gcaattattt cctgcctcca ggagccgtta ggtactgggg ccaggggact 360 caggtcacag taagcagc 378 <210> 125 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 125 caggtgcagc tccaggagag cggtggcgga tcagtgcagg ctggaggctc cctcagactg 60 tcctgcaccg tgagccgcta taccgcctcc gtcaactata tgggatggtt taggcaggct 120 ccgggcaagg agcgcgaggg ggtcgcgact atcttcaccg gagccggtac tacctattac 180 gctaattctg ttaaaggccg ctttaccatt agtcgcgaca acgctaagaa cacagcttac 240 ctccagatga actctctgaa gccagaggat accgccatgt attactgcgc cgtggacttc 300 cggggcggtt tgctctaccg cccggcctac gaatacacct atcgcggcca gggcacgcag 360 gtcacggtgt cctca 375 <210> 126 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 126 caggtgcagc tccaagagtc cggtggaggc agcgtccagg ccgggggtag tcttaggctc 60 agctgtgctg ccagtggaga cacctactct tcctattgca tgggatggtt cagacaggcc 120 cccggcaaag agcgcgaggg cgttgcattc atcgactccg acggctccac tcgctacgcc 180 gatagcgtgg agggccgttt taccatctcc aaggacaacg cgaagaacac tctgtatctg 240 caaatgaact ccctgaagcc cgaagacacc gccatgtact attgcgcggc tgagccatac 300 tgtagtggcg gatatcatcg taaggaaatg gcagagttcg gctattgggg ccagggcacc 360 caggtcactg tgagttcc 378 <210> 127 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 127 caggtgcagt tgcaggaatc cggcggaggc tctgtgcagg cgggcggttc cctccgcctg 60 agttgtgccg cgtctggcta tacttactct tcctattgta tgggatggtt ccggcaagcg 120 cccggcaaag agcgggaggg cgttgcgcat atcgacagtg atggtagcac cagttacgct 180 gatagcgtga aaggcagatt cactatctca aaggataacg cgaagaacac tctttacctc 240 cagatgaact cccttaaacc tgaggatacc gcgatgtatt actgtgctgc cgaccccatt 300 cccggccctg gatactgtga cggaggccct aacaagtacc gtgggcaagg aacacaggtc 360 acagtgtcca gc 372 <210> 128 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 128 caggtgcaac tccaggagtc tggcgggggc agcgtccagg caggtggaag tctccgtctc 60 tcatgtgctg ccagcggcta tacatactcc agctactgta tgggatggtt tagacaggca 120 cccggcaagg agcgcgaagg ggtggcccat atcgactccg atggcagcac aacctatgcc 180 gactctgtga aagggcggtt cgccatctcc aaggacaacg ctaagaatac cctgtacctc 240 cagatgaact ctctgaagcc tgaggacacc gccatgtatt actgcgctgc cgacccaatc 300 cctggcccag gttactgcga tgggggacca aacaaatatt ggggacaggg cacgcaggtt 360 acagtctcca gc 372 <210> 129 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 129 caggtccaac tccaggaaag tggaggtggc tctgttcagg ccgggggcag cctgaggctg 60 agctgcaccg gctcaggcta tacagccagt aataactgca tgggctggtt ccgtcaagcg 120 cccggcaaag agcgtgaagg tgtggccgta atttttaccg gcgctggcac cagctattac 180 gacagttccg tgggccgtct gttcatcagc tcacaggacg ccgcttccac cctcgatcag 240 ttgctgatga gccttctgcc cgatgacacc gcagtaatgt actgtggagc cgaagatgac 300 tgcacactgc tcctgatgac gccaaacccc gatgaccaat ggtcccgcct gagtgtgtcc 360 tcc 363 <210> 130 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 130 caggtgcagc tccaggagag cgggggcggt tctgttcagg cgggaggcag cctgcgtctg 60 tcctgtgcag cctctggtta cacagacagt cgttactgca tgggctggtt ccgcaaggca 120 cctggaaagg agcgcgaggg tgttgcgcac atcgactccg acgggagcac tagctatgct 180 gacagcgtga aggggcgctt cactatcagc aaggataacg cgaaaaacac cttgtacctt 240 cagatgaact ccctcaaacc cgaagacaca gcgatgtact attgtgccgc tgatccgatc 300 ccagggcctg gctactgtga tggtggacct aataagtact gggggcaggg aactcaggtg 360 accgtgtcat ca 372 <210> 131 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 131 caggtccagt tgcaggaatc tggaggcggt tccgtgcaag cagggggctc actcagactg 60 tcctgcgctg ccagcggcta cacttactct tcatattgca tgggctggtt ccgccaggca 120 ccgggcaagg agcgggaagg cgtggccgct attgatagcg atggctctac gcgctacgca 180 gatagcgtga aagggaggtt cacgatctcc aaagataatg ccaagaaaat tctgtatctc 240 cagatgaact ctctgaaggt cgaggacacc gccatgtact attgtgcagc cgaaccctac 300 tgttctggtg gctacaagag gactatggtg gccgagttcg gcttctgggg ccaggggacc 360 caagtgactg tcagtagc 378 <210> 132 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 132 caggtgcaac ttcaggagag cggtggcgga tctgtgcagg ctggagggtc tctgaagctg 60 tcctgcgcgg ccagcggtta cacatacagt agctactgca tgggatggtt tcgtcaggcc 120 ccaggcaagg agcgcgaagg agtggcgcac atcgactccg atgggtccac cacatacgcc 180 gactccgtga agggccgttt cacaatcagc aaggataacg cgaagaacac gctgtacttg 240 cagatgaact ctctcaaacc agaggacact gcaatgtact attgcgcggc tgaccccatc 300 cctggccctg gttactgtga cggtggcccc aacaattact gggggcaagg gacccaagtc 360 accgtgtcct cc 372 <210> 133 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 133 caggtccagc tccaggagtc cggcgggggc tccgtccagg cagggggctc cctgcgtctg 60 tcatgcgccg cttctgggta tacctacagt tcctattgta tgggttggtt tcgccaagca 120 cccggtaagg agcgcgaagg tattgcgcac attgatagcg atggctccac aacctatgct 180 gacagtgtga aaggacgctt cactatttcc aaggataacg ctaagaacac actctacctt 240 cagatgaaca gcctgaagcc ggaagacacc gcaatgtact attgtgcagc tgaccccatt 300 cctggacccg gttactgtga tggaggtcct aataactatt ggggacaggg cactcaagtg 360 accgtctcaa gc 372 <210> 134 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 134 caggtgcagt tgcaggagag cgggggtggc tctgtgcagg ccgggggctc cctgaggctg 60 agctgcgcgg ccagcgggta cagctactct agctattgca tgggttggtt ccgccaggcc 120 cctggcaagg agcgcgaggg agtggccacg attgactcag atggcatgac ccgttatgcg 180 gattccgtca aggggcgctt caccatcagc aaagataacg ccaaaaatac cctgtacttg 240 cagatgaact cactgaaacc tgaggataca gccatgtatt actgcgcagc tccgctctat 300 gactgtgact ctggtgccgt gggtagaaac ccaccttact gggggcaggg aacccaggtg 360 accgtgtcct ca 372 <210> 135 <211> 366 <212> DNA <213> Artificial Sequence ...

Claims

1. An IL10Rb binding molecule that specifically binds to the extracellular domain of IL10Rb, Contains single-domain antibodies (sdAb), sdAb is shown in the following table: As shown in the row, the complementarity determination regions include Complementarity Determination Region 1 (CDR1), CDR2, and CDR3, IL10Rb binding molecule.

2. The IL10Rb-binding molecule according to claim 1, wherein the sdAb has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one polypeptide sequence of SEQ ID NO: 61, 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, and 105.

3. The IL10Rb binding molecule according to claim 2, wherein sdAb comprises a sequence selected from the group consisting of SEQ ID NO: 61, 9, 101, 1, 45, 57, and 105.

4. The IL10Rb binding molecule according to claim 3, wherein sdAb further comprises an amino acid substitution Q1E.

5. The IL10Rb-binding molecule according to claim 2, wherein the sdAb has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% identity with the polypeptide having the sequence shown in SEQ ID NO:

61.

6. An IL10Rb-binding molecule according to any one of claims 1 to 5, comprising a humanized or otherwise CDR grafted onto a heterogeneous framework.

7. An IL10Rb-binding molecule according to any one of claims 1 to 5, further comprising a labeling agent, an imaging agent, and / or a therapeutic agent.

8. A pharmaceutical composition for treating or preventing a disease, disorder, or condition in a mammalian subject, comprising a therapeutically effective amount of an IL10Rb binding molecule according to any one of claims 1 to 5 or a pharmaceutically acceptable formulation thereof.

9. An IL10Rb-binding molecule according to any one of claims 1 to 5, for use in the isolation, depletion, or enrichment of IL10Rb+ cells in a biological sample.

10. A nucleic acid encoding an IL10Rb-binding molecule according to any one of claims 1 to 5.

11. A recombinant virus or nonviral vector comprising the nucleic acid described in claim 10.

12. A host cell comprising the nucleic acid described in claim 10.

13. A pharmaceutical formulation comprising the virus or nonviral vector according to claim 11.

14. A kit comprising an IL10Rb-binding molecule according to any one of claims 1 to 5.

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