IL27Rα-binding molecule and method of use

Abstract

To provide biologically active molecules comprising a single domain antibody (sdAb) that specifically binds to the extracellular domain of human IL27Rα, compositions comprising such antibodies, and methods of use thereof.SOLUTION: An IL27Rα-binding molecule is provided, which comprises a single domain antibody (sdAb) that specifically binds to the extracellular domain of IL2Rb and has a specific amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 061,562, filed on August 5, 2020; U.S. Provisional Application No. 63 / 078,745, filed on September 15, 2020; and U.S. Provisional Application No. 63 / 135,884, filed on January 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 the IL27 receptor α (IL27Rα), compositions comprising such single - domain antibodies, and methods of using the same.

Background Art

[0003] Background The cytokine IL - 27 is a heterodimeric cytokine consisting of p28 and EBI3, two non - covalently associated subunits. The p28 subunit belongs to the four - helix - bundle cytokine family, while EBI3 is the shortest form of a soluble cytokine receptor having two representative cytokine - binding domains (Pflanz S, et al., Immunity. 2002 Jun;16(6):779 - 90).

[0004] The interleukin-27 receptor (IL27R) is a type I cytokine receptor for interleukin-27 (IL27). IL27R is a heterodimer composed of the IL27Rα subunit and glycoprotein 130 (IL6Rb). IL27 is expressed by antigen-presenting cells and induces differentiation of diverse T cell populations in the immune system. When IL27 binds to IL27R, intracellular signaling is initiated via several Jak family kinases that induce phosphorylation of STAT1 and STAT3. In activated T cells, IL27 primarily signals via STAT3

[23] , whereas in memory B cells, it primarily signals via STAT3. IL27 has been shown to possess both pro-inflammatory and anti-inflammatory properties, and this pro-inflammatory or anti-inflammatory response is influenced by the state of IL27R-expressing cells.

[0005] The IL27Rα subunit (also known as the TCCR- or WSX-1 receptor) is an intellectual property-protected IL27 receptor subunit. Mature (signal peptide-less) IL27Rα is a 604-amino acid polypeptide with a 484-amino acid extracellular domain. The extracellular domain of IL27Rα contains five domains: D1-D5. D1 and D2 are primary cytokine-binding domains, while the fibronectin type III (Fn3) domains D3, D4, and D5 are involved in ligand recognition to a considerably lesser extent than D1 and D2. Based on structural analysis, the Fn3 domains do not contribute to binding in the complex when the IL27 ligand is bound. Domains D1 and D2 are highly conserved, but the sequence of the Fn3 domain is more variable.

[0006] IL27 exhibits high (nanomolar) affinity for the IL27Ra subunit. The [IL27 / IL27Rα] complex associates with IL27Ra to complete the IL27 receptor signaling complex. The binding of gp130 to the [IL27 / IL27Rα] complex is considerably weaker than the interaction between IL-27 and IL-27R, which is somewhat common for cytokine receptor subunits (Pflanz S, et al., J Immunol. 2004 Feb 15;172(4):2225-31). The D5 domain of IL-27R and the D6 domain of gp130 come together in close proximity in the membrane for each receptor to assume a "C" shape. This is necessary for the receptor complex to induce JAK binding in the intracellular domains of both receptors.

[0007] Monoclonal antibodies are the most widely used reagents for protein detection and quantification; however, monoclonal antibodies are large molecules of approximately 150 kDa, and their size can potentially 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 present 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 IL27Ra.

[0009] This disclosure provides a polypeptide that specifically binds to the extracellular domain of IL27Ra.

[0010] This disclosure provides an IL27Ra-binding molecule that specifically binds to the extracellular domain of human IL27Ra (hIL27Ra).

[0011] In some embodiments, the IL27Ra-binding molecule includes a single-domain antibody (sdAb) that specifically binds to the extracellular domain of human IL27Ra.

[0012] In some embodiments, the IL27Ra-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.

[0013] In some embodiments, the IL27Ra-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.

[0014] In some embodiments, the IL27Ra-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 conservative amino acid substitutions) or 100% identity with respect to any single polypeptide sequence of SEQ ID NO: 2 to 25, as shown in Table 1 below.

[0015] (Table 1) TIFF0007894676000001.tif60160TIFF0007894676000002.tif235160TIFF0007894676000003.tif235160TIFF0007894676000004.tif204160

[0016] In some embodiments, the aforementioned set of CDRs are incorporated into a humanized VHH framework to form a "humanized" sdAb IL27Ra-binding molecule.

[0017] Furthermore, this disclosure provides a chemical or recombinant process method for preparing the IL27Ra-bound molecule of this disclosure.

[0018] Furthermore, this disclosure provides nucleic acids encoding IL27Ra-binding molecules. Table 2 below shows examples of DNA sequences encoding IL27Ra-binding molecules as described herein.

[0019] (Table 2) DNA sequences encoding VHH in Table 1 TIFF0007894676000005.tif202158TIFF0007894676000006.tif202158TIFF00078946760 00007.tif202158TIFF0007894676000008.tif202158TIFF0007894676000009.tif163158

[0020] In some embodiments, IL27Ra is the mouse IL27Ra.

[0021] In some embodiments, the IL27Ra-binding molecule includes a single-domain antibody (sdAb) that specifically binds to the extracellular domain of mouse or rat IL27Ra (mIL27Ra).

[0022] In some embodiments, the IL27Ra-binding molecule is an sdAb, which comprises a set of CDRs corresponding to CDR1, CDR2, and CDR3, as shown in the row in Table 3 below.

[0023] In some embodiments, the IL27Ra-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.

[0024] In some embodiments, the IL27Ra-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 any one polypeptide sequence of SEQ ID NO: 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, and 174, as shown in Table 3 below.

[0025] (Table 3) mMIL2Rg VHH and CDR amino acid (AA) sequences TIFF0007894676000010.tif135167TIFF0007894676000011.tif216167TIFF0007894676000012.tif216167TIFF0007894676000013.tif54167

[0026] In some embodiments, the aforementioned set of CDRs are incorporated into a humanized VHH framework to form a "humanized" sdAb IL27Rα-binding molecule.

[0027] Furthermore, this disclosure provides a chemical or recombinant process method for preparing the IL27Rα-binding molecule of this disclosure.

[0028] Furthermore, this disclosure provides nucleic acids encoding IL27Ra-binding molecules. Table 4 below shows an example of a DNA sequence encoding an hIL27Ra-binding molecule, as described in Table 3 above.

[0029] (Table 4) DNA sequences encoding VHH in Table 3 TIFF0007894676000014.tif91164TIFF0007894676000015.tif212164TIFF0007894676000016.tif212164TIFF0007894676000017.tif91164

[0030] Furthermore, the Disclosure provides recombinant viral vectors and nonviral vectors comprising the IL27Rα-binding molecule of the Disclosure or a nucleic acid encoding the CDR of the IL27Rα-binding molecule of the Disclosure.

[0031] Furthermore, the Disclosure provides host cells comprising recombinant viral vectors and non-viral vectors, each comprising a nucleic acid encoding the IL27Rα-binding molecule of the Disclosure or the CDR of the IL27Rα-binding molecule of the Disclosure.

[0032] Furthermore, the Disclosure provides host cells comprising recombinant viral vectors and non-viral vectors, each comprising a nucleic acid encoding the IL27Rα-binding molecule of the Disclosure or the CDR of the IL27Rα-binding molecule of the Disclosure.

[0033] Furthermore, this disclosure provides a kit containing the IL27Rα-binding molecule of this disclosure.

[0034] In another aspect, the Disclosure provides constructs for identifying IL27Rα-expressing cells, wherein an IL27Rα-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 IL27Rα-expressing cells in a subject, comprising the steps of administering an effective amount of an IL27Rα-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 IL27Rα-binding molecule.

[0035] In another aspect, the present disclosure provides an IL27Rα-binding molecule modified to extend the duration of action in vivo, wherein the IL27Rα-binding molecule is conjugated to one or more carrier molecules.

[0036] This disclosure provides an IL27Rα-binding molecule comprising a polypeptide sequence that specifically binds to the extracellular domain of IL27Rα, and a method for using it in the isolation, depletion, or enrichment of IL27Rα-expressing cells in a biological sample. [Invention 1001] An IL27Rα-binding molecule that specifically binds to the extracellular domain of IL2Rb. [Invention 1002] The IL27Rα binding molecule of the present invention 1001, wherein the IL2Rb binding molecule contains a single-domain antibody (sdAb). [Invention 1003] sdAb is shown in the following table: The IL27Rα-binding molecule of the present invention 1002, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3, as shown in the row of TIFF0007894676000018.tif177167TIFF0007894676000019.tif151167. [Invention 1004] An IL27Rα-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: 2 to 25. [Invention 1005] sdAb is shown in the following table: The IL27Rα-binding molecule of the present invention 1002, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3, as shown in the row of TIFF0007894676000020.tif84170. [Invention 1006] An IL27Rα-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: 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, and 174. [Invention 1007] An IL27Rα-binding molecule of either Invention 1003 or 1005, comprising a humanized sdAb or a CDR grafted onto a heterogeneous framework. [Invention 1008] An IL27Rα-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] An IL27Rα-binding molecule according to any of invention 1001-1008 for use in the isolation, depletion, or enrichment of IL27Rα+ cells from a biological sample. [Invention 1011] A nucleic acid sequence encoding any of the IL27Rα-binding molecules of the present invention 1001 to 1008. [Invention 1012] A recombinant viral vector 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 kit containing any of the IL2Rb-binding molecules described in invention 1001 to 1008. [Modes for carrying out the invention]

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] (Table 5) Abbreviations of amino acids TIFF0007894676000021.tif110128

[0045] 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).

[0046] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0047] 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.

[0048] 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.

[0049] 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 IL27Rα-binding molecule or engineered cells expressing an IL27Rα-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.

[0050] 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 It is measured by [method].

[0051] 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.

[0052] 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.

[0053] antibody: As used herein, the term “antibody” encompasses a broad range of immunoglobulin derivatives, including but not limited to (a) glycosylated or non-glycosylated immunoglobulins that specifically bind to a target molecule, and (b) antibody fragments such as single-domain antibodies. 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 mice, humans, horses, camelids, and cartilaginous fish, including but not limited to sharks. The term “antibody” encompasses 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.

[0054] 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.

[0055] IL27Rα cells: The terms “IL27Rα cells,” “IL27Rα-expressing cells,” “IL27Rα-positive cells,” and “IL27Rα+” cells are used herein synonymously to refer to cells that express and show the IL27Rα antigen on the extracellular surface of the cell membrane. Similarly, the terms “IL27Rα-negative cells” and “IL27Rα- cells” are used herein synonymously to describe cells that do not express or show the IL27Rα antigen on their cell surface.

[0056] 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 variant thereof is intended to be within the scope of the terms as defined and used herein. In the context of this disclosure, unless otherwise specified, the numbering of CDR locations is according to Kabat, or a hybrid of Kabat and Chothia numbering rules.

[0057] 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%.

[0058] 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.

[0059] 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.

[0060] 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 (about 50%) change 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.

[0061] 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 higher concentration (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 existing after administration), or (b) the molecule is present at a higher concentration than the environment in which it was made (e.g., a recombinantly modified bacterial or mammalian cell).

[0062] 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 of the plasma membrane of a cell. The cell surface protein may be a transmembrane protein, a cell surface protein, or a membrane-bound protein.

[0063] 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).

[0064] 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.

[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] 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.

[0077] 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.

[0078] 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.

[0079] 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."

[0080] 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.

[0081] 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 in a binding pair is said to specifically bind to its second molecule if it does not bind in significant amounts to other components present in the sample. A first molecule in a binding pair is said to specifically bind to its second molecule if 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, if the first molecule in a binding pair is an antibody, the equilibrium dissociation constant between the antibody and antigen is approximately 10 when determined, for example, by scatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239). 6 Over M, or about 10 8 Over M, or about 10 10 Over M, or about 10 11 Over M, about 10 12 If the M value is greater 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 IL27Rα-binding sdAb and the receptor contains IL27Rα, the equilibrium dissociation constant of IL27Rα-binding sdAb / IL27RαECD is approximately 10 5 Over M, or about 10 6 Over M, or about 10 7Over M, or about 10 8 Over M, or about 10 9 Over M, or about 10 10 M or more, or about 10 11If the M value is greater than M, IL27Rα-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, scatcharded 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 IL27Rα isoforms (e.g., IL27Rα-binding sdAb). The binding affinity of IL27Rα-binding molecules to IL27Rα as used herein may be determined and / or quantified by surface plasmon resonance ("SPR"). When evaluating the binding affinity of IL27Rα-binding molecules to IL27Rα, one member of the binding pair may be immobilized, while the other element of the binding pair is provided in the mobile phase.In some embodiments, a sensor chip on which a 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 with NTA by incorporating a chelated peptide containing a polyhistidine sequence (e.g., 6xHis (SEQ ID NO: 195) or 8xHis (SEQ ID NO: 196)) for retention on the chip. In some embodiments, the IL27Rα-binding molecule may be immobilized on the chip, and IL27Rα (or its ECD fragment) may be provided in the mobile phase. Alternatively, IL27Rα (or its ECD fragment) may be immobilized on the chip, and the IL27Rα-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 proteins to be evaluated.Alternatively, when evaluating the binding affinity of an IL27Rα-binding molecule to IL27Rα using SPR, the IL2Rb-binding molecule may be derivatized by C-terminal addition of a polyHis sequence (e.g., 6xHis (SEQ ID NO: 195) or 8xHis (SEQ ID NO: 196)) and immobilized on an NTA derivatized sensor chip, and the IL27Rα receptor subunit whose ligand binding affinity is being evaluated is provided in the mobile phase. Means for incorporating a polyHis sequence into the C-terminus of an IL27Rα-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 IL27Rα-binding molecule to IL27Rα is evaluated using SPR in general accordance with the disclosure of examples.

[0082] 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.

[0083] 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.

[0084] 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 the IL27Rα isoform, referred to synonymously with IL27Rα cells, IL27Rα+ cells, IL27Rα T cells, or IL27Rα+ T cells.

[0085] 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.

[0086] 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.

[0087] 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 IL27Rα-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).

[0088] 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 those mentioned above. + In this specification, the term Treg is 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.

[0089] 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.

[0090] Wild type: In this specification, “wild-type,” “WT,” or “native” means the naturally occurring amino acid or nucleotide sequence, including allelic variations. Wild-type proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., have an amino acid or nucleotide sequence that has not been modified by human intervention.

[0091] IL27Rα The IL27Rα-binding molecule of this disclosure specifically binds to the extracellular domain of IL27Rα.

[0092] Human IL27Rα In one embodiment, it specifically binds to the extracellular domain of the human IL27Rα receptor subunit (hIL27Rα). hIL27Rα contains a 32-amino acid N-terminal signal sequence, and is expressed as a 636-amino acid precursor that undergoes post-translational cleavage of the N-terminal signal sequence to become a 604-amino acid mature protein. The classic full-long acid hIL27Rα precursor (including the signal peptide) has the following amino acid sequence: It is a 636-amino acid polypeptide containing TIFF0007894676000022.tif62134.

[0093] For the purposes of this disclosure, the amino acid residue numbering of the human IL27Rα polypeptide described herein follows the numbering of this classical sequence (UniProt reference number Q6UWB1, SEQ ID NO:1). Amino acids 1-32 of SEQ ID NO:1 have been identified as the signal peptide of hIL27Rα, amino acids 33-516 of SEQ ID NO:1 as the extracellular domain, amino acids 517-537 of SEQ ID NO:1 as the transmembrane domain, and amino acids 538-636 of SEQ ID NO:1 as the intracellular domain.

[0094] For the purpose of producing antibodies that bind to the ECD of IL27Rα, immunization can be performed using the extracellular domain of hIL27Rα. The extracellular domain of hIL27Rα has the following sequence: TIFF0007894676000023.tif49134 is a 484-amino acid polypeptide.

[0095] Mouse IL27Rα In one embodiment, it specifically binds to the extracellular domain of the mouse or rat IL27Rα receptor subunit (mIL27Rα). mIL27Rα contains a 24-amino acid N-terminal signal sequence and is expressed as a 623-amino acid precursor that is post-translationally cleaved to form a 599-amino acid mature protein. The classic full-long acid mIL27Rα precursor (containing the 24-signal peptide) has the following amino acid sequence: This is a 623-amino acid polypeptide containing TIFF0007894676000024.tif62134.

[0096] For the purposes of this disclosure, the amino acid residue numbering of the mRNA polypeptide as described herein follows the numbering of this classical sequence (UniProt reference number O70394, SEQ ID NO: 193). Amino acids 1-24 of SEQ ID NO: 193 have been identified as the mRNA signal peptide, amino acids 23-510 of SEQ ID NO: 193 as the extracellular domain, amino acids 511-531 of SEQ ID NO: 193 as the transmembrane domain, and amino acids 532-623 of SEQ ID NO: 193 as the intracellular domain.

[0097] Immunization can be performed using the extracellular domain of mIL27Rα for the purpose of producing antibodies that bind to the ECD of IL27Rα. The extracellular domain of the mIL27Rα receptor has the following sequence: TIFF0007894676000025.tif49133 is a 486-amino acid polypeptide.

[0098] IL27Rα-binding molecule and single-domain antibody In some embodiments, the IL27Rα-binding molecule of this disclosure is a single-domain antibody (sdAb). This disclosure relates to an IL27Rα-binding molecule, including a single-domain antibody (sdAb), which specifically binds to the extracellular domain of human IL27Rα isoforms (hIL27Rα) found on all IL27Rα-expressing cells.

[0099] 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. hIL27Rα-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 hIL27Rα or an immunologically active fragment thereof. 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 Seen in (Basel) 8(1). Alternatively, hIL27Rα 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 hIL27Rα or an immunologically active fragment thereof. hIL27Rα-conjugated sdAbs may also be obtained by splitting the dimeric variable domain derived from immunoglobulin G (IgG) isotypes from other mammalian species, including humans, rats, and rabbits, immunized with the extracellular domain of hIL27Rα or an immunologically active fragment thereof. Currently, most sdAb studies are based on heavy-chain variable domains, but sdAbs derived from light chains have also been shown to specifically bind to target proteins containing antigenic immunization sequences. Moller et al., J Biol Chem. 285(49):38348-38361, 2010.

[0100] In some embodiments, the sdAb is a VHH. A VHH is a type of sdAb having a single monomeric weight-chain variable antibody domain. Like conventional antibodies, VHHs can specifically bind to specific antigens. Exemplary VHHs have a molecular weight of approximately 12–15 kDa, which is considerably smaller than conventional mammalian antibodies (150–160 kDa) composed of two heavy chains and two light chains. VHHs may be found in nature in camelid mammals that lack light chains (e.g., camels, llamas, dromedaries, alpacas, and guanacos), or they may be produced from them.

[0101] This disclosure provides an IL27Rα-binding molecule containing a polypeptide having at least 75%, 80%, 90%, 95%, 98%, 99%, or 100% identity with any one polypeptide of SEQ ID NO:2 to 25.

[0102] This disclosure provides an IL27Rα-binding molecule containing a polypeptide having at least 75%, 80%, 90%, 95%, 98%, 99%, or 100% identity with any one polypeptide of SEQ ID NO: 61-74.

[0103] This disclosure provides IL27Rα-binding molecules comprising CDR1, CDR2, and CDR3 as described in the rows of Table 1 provided herein. In some embodiments, CDR1, CDR2, and CDR3 may each independently have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with respect to the sequences described in the rows of Table 1 provided herein, and may have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

[0104] This disclosure provides IL27Rα-binding molecules comprising CDR1, CDR2, and CDR3 as described in the rows of Table 3 provided herein. In some embodiments, CDR1, CDR2, and CDR3 may each independently have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequences described in the rows of Table 3 provided herein, and may have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

[0105] experiment The single-domain antibody of this disclosure was obtained from camels by immunization with the extracellular domain of the IL27Rα receptor. The IL27RαVHH molecule of this disclosure was constructed largely in accordance with the examples disclosed. Briefly, camels were successively immunized over several weeks with a subcutaneous adjuvant composition containing a fusion protein comprising the extracellular domain of IL27Rα, the human IgG1 hinge domain, and the human IgG1 heavy chain Fc, produced by recombinant using human IL27Rα and mouse IL27Rα ECDs. After immunization, RNA extracted from blood samples of appropriate size VHH-hinge-CH2-CH3 was transcribed to produce DNA sequences, digested, and isolated to identify a fragment of approximately 400 bp containing the nucleic acid sequence encoding the VHH domain. To facilitate insertion into phagemide vectors in frame with the sequence encoding the his-tag, isolated sequences were digested with restriction endonucleases and introduced into Escherichia coli (E. coli) by transformation to create a phage library. Multiple phage library biopannings were performed to identify VHHs bound to the ECD of IL27Rα (human or mouse, as appropriate). Periplasmic extract ELISA (PE-ELISA) in 96-well plates and individual phage clones were isolated for selective binding confirmed by colorimetric analysis. IL27Rα-binding molecules showing specific binding to the IL27Rα antigen were isolated, sequenced, and sequence-analyzed to identify the VHH sequence, CDR, 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 within a specific collection of antigen-binding molecules belonging to the same germline family, having the same CDR3 length and exhibiting 70% or greater homology in the CDR3 sequence. Table 1 shows VHH molecules that specifically bound to the hIL27RαECD antigen (anti-human IL27RαVHH) and CDRs isolated from such VHH. Table 3 shows VHH molecules that specifically bound to the mIL27RαECD antigen (anti-mouse IL27RαVHH) and CDRs isolated from such VHH.The nucleic acid sequences encoding VHH in Tables 1 and 3 are shown in Tables 2 and 4, respectively.

[0106] To further fully characterize the binding properties of the VHH molecules prepared according to the above description and to evaluate their binding affinity, representative examples of each human VHH chronotype were subjected to surface plasmon resonance (SPR) analysis, largely in accordance with the disclosure in Example 5 of this specification. The results of these SPR studies are summarized in Table 6 below.

[0107] (Table 6) Binding of anti-hIL27Ra monoFc VHH (ligand) to hIL27Ra-his (Antigen: Origene, Catalog number TP307012) TIFF0007894676000026.tif54168 * Both the association rate constant and the dissociation rate constant may be suppressed when Rmax > 100. If this effect exists, it is likely to be offset by the reaction rate ratio, i.e., the affinity constant.

[0108] As demonstrated by the data shown in Table 6 above, the IL27RαVHH binding molecule exhibited specific binding to the antigen and showed a certain range of affinity for the IL27Rα antigen.

[0109] In some cases, due to sequence or structural similarities between the extracellular domains of IL27Rα receptors from various mammalian species, immunization with an antigen derived from IL27Rα of a first mammalian species (e.g., hIL27Rα-ECD) may produce antibodies that specifically bind to IL27Rα receptors of one or more further mammalian species. Such antibodies are called "cross-reactive." For example, immunization of a camelid animal with a human-derived antigen (e.g., hIL27Rα-ECD) may produce antibodies that cross-reactive to both mouse and human receptors. The evaluation of cross-reactivity of antibodies to receptors from other mammalian species can be readily determined by those skilled in the art using methods related to the evaluation of binding affinity and / or specific binding, as described elsewhere herein, for example, flow cytometry or SPR. Consequently, the use of the terms "human IL27RαVHH" or "hIL27RαVHH" merely indicates that the species of IL27Rα antigen used to immunize the camelid animals from which VHH originated was human IL27Rα (e.g., hIL27Rα, ECD, SEQ ID NO: 192), and should not be understood as a limitation regarding the specific binding affinity of VHH to hIL27Rα molecules of other mammalian species. Similarly, the use of the terms "mouse IL27RαVHH" or "mIL27Rα" merely indicates that the species of IL27Rα antigen used to immunize the camelid animals from which VHH originated was mouse IL27Rα (e.g., mIL27RαECD, SEQ ID NO: 194), and should not be understood as a limitation regarding the specific binding affinity of VHH to IL27Rα molecules of other mammalian species.

[0110] Modified single-domain antibodies CDR graft sdAb In some embodiments, the IL27Rα-conjugated sdAb of this disclosure is a CDR-grafted IL27Rα-conjugated sdAb. To prepare the CDR-grafted sdAb, CDRs obtained from antibodies, heavy chain antibodies, and sdAbs derived therefrom may be grafted onto another framework as described in Saerens, et al. (2005) J. Mol Biol 352:597-607. In some embodiments, this disclosure provides an IL27Rα-conjugated molecule comprising a CDR-grafted IL27Rα-conjugated sdAb, the CDR-grafted IL27Rα-conjugated sdAb comprising a set of CDRs 1, 2, and 3 as shown in the row in Table 3 above.

[0111] Chimeric sdAb and humanized sdAb Any framework region can be used with the CDR as described herein. In some embodiments, the IL27Rα-conjugated sdAb is a chimeric sdAb, in which the CDR originates from one species (e.g., camel) and the framework and / or constant region originates from another species (e.g., human or mouse). In certain embodiments, the framework region is a human or humanized sequence. Thus, a humanized IL27Rα-conjugated sdAb derived from hIL27Rα-conjugated VHH is considered to be within the scope of this disclosure. Techniques for humanizing single-domain antibodies of camelid animals 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.

[0112] In some embodiments, V as described herein H H can be humanized to include the human framework domain. Humanized V HExamples 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).

[0113] IL27Rα-binding molecule containing further agents In some embodiments, the IL27Rα-binding molecules of the present disclosure include an IL27Rα single-domain antibody (sdAb) conjugated to one or more further biologically active agents, including but not limited to a combination of therapeutic agents, chemically active agents, optically active agents, or radioactive materials. The 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 IL27Rα-binding sdAb, and this combination results in an IL27Rα-binding molecule with further utility or different utility.

[0114] 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 the IL27Rα-binding molecule to a specific cell or tissue type; therapeutic agents (e.g., therapeutic agents containing small molecules or polypeptides); 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 IL27Rα-binding molecule facilitates the release of the therapeutic agent into the intracellular cytoplasm during the internal translocation of the IL27Rα-binding molecule. Using an uncleavable linker would allow for the release of the IL27Rα-binding molecule during digestion. Alternatively, an uncleavable linker could be used in conjunction with agents that do not require release from the antibody (e.g., imaging agents).

[0115] In some embodiments, the IL27Rα-binding molecule comprises an IL27Rα-bonded sdAb that is stably bound to a further agent, which is linked via a linker. The linker is a covalent bond between the two elements of the IL27Rα-binding molecule (e.g., the hIL27Rα-bonded VHH and the 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 IL27Rα-bonded sdAb and the linked agent. Examples of chemical linkers include arylacetylenes, ethylene glycol oligomers containing 2 to 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. Multimers 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 to provide a mobile linker that can be used to conjugate heterogeneous amino acid sequences to the IL27Rα-linked sdAb disclosed herein. In some embodiments, the linker has the formula (GGGS)n(SEQ ID NO:197), (GGGSG)n(SEQ ID NO:198), (GGGGS)n(SEQ ID NO:199), (GGS)nG(SEQ ID NO:200), or (GGSG)n(SEQ ID NO:201), where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0116] Immunomodulators In some embodiments, the IL27Rα-binding molecule of the Disclosure is functionally conjugated to an immunomodulator (immunoconjugate). Immunomodulators that can be conjugated to the hIL27Rα-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 IL27Rα or cells expressing IL27Rα.

[0117] Flag tag In some embodiments, the IL27Rα-binding molecule of this disclosure is 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 IL27Rα-binding sdAb polypeptide further comprises a C-terminal c-myc epitope tag.

[0118] Chelated peptides In some embodiments, the IL27Rα-binding molecule of this disclosure is functionally linked to one or more transition metal chelate polypeptide sequences. Incorporating such a transition metal chelate domain facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Patent No. 4,569,794 by Smith et al., issued February 11, 1986. Examples of transition metal chelate polypeptides useful in the implementation of this IL27Rα-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. These full disclosures are incorporated herein by reference. Specific transition metal chelate polypeptides useful in the implementation of this IL27Rα-binding molecule are polypeptides containing 3 to 6 consecutive histidine residues (SEQ ID NO: 202), such as 6-histidine (His)6 peptide (SEQ ID NO: 195), often referred to in the art as "His tags." In general, in accordance with the disclosures 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 an hIL27Rα-binding molecule to such a chelated peptide facilitates the targeted delivery of transition metal ions to IL27Rα-expressing cells as a kinetically inert or kinetically unstable complex, in addition to providing a purification "handle" for recombinant proteins or facilitating immobilization on an SPR sensor chip. The transition metal ion is a reporter molecule, e.g., a fluorescent compound or radioimaging agent, and includes radioactive materials or therapeutic agents.

[0119] Carrier molecule In some embodiments, the IL27Rα-bound sdAb of this disclosure may be conjugated with 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 IL27Rα-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.

[0120] Water-soluble polymers In some embodiments, the IL27Rα-linked sdAb is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the implementation of this IL27Rα-linked molecule include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohol, polysaccharides, polyα-hydroxy acids, polyvinyl alcohol (PVA), polyphosphoren, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0121] 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 1, 2, 3 (or more) PEG moieties are each attached to the polypeptide. In some embodiments, PEG may be directly or covalently attached to the sdAb (e.g., via a lysine side chain, a sulfhydryl group of cysteine, or an N-terminal amine), and optionally, a linker may be used between the PEG and the sdAb. In some embodiments, the IL27Rα-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.

[0122] In some cases, the IL27Rα-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 IL27Rα-binding molecules of this disclosure, particularly when aldehyde chemistry is used, position 1 of the IL27Rα-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 IL27Rα-binding molecule of this disclosure includes an amino acid substitution selected from the group of Q1E and Q1D.

[0123] PEGs suitable for conjugation into polypeptide sequences are generally soluble in water at room temperature and have a 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.

[0124] The molecular weight of PEG used in the IL27Rα-binding molecule is not limited to any particular range. The molecular weight of the PEG component of the IL27Rα-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 IL27Rα-binding molecule, the PEG is a 40kD branched PEG containing two 20kD arms.

[0125] This disclosure also intends IL27Rα-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 IL27Rα-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.

[0126] 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.

[0127] 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.

[0128] PEG can be bound to the IL27Rα-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.

[0129] 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.

[0130] The PEG portion of the PEGylated IL27Rα-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.

[0131] 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 a cysteine ​​residue at position S354 on one chain and at Y349 on the other chain, resulting in the formation of a stabilizing disulfide bond between the two antibody heavy chains in the Fc 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., IL27Rα-binding sdAb) on a first Fc monomer having a "knob" modification, and a second polypeptide on a second Fc monomer having a "hole" modification.

[0132] Targeted domains In some embodiments, the IL27Rα-binding molecule is provided as a component of a polyvalent (e.g., bivalent) fusion protein having such a targeting domain, optionally incorporating a linker between the IL27Rα-binding sdAb sequence of the fusion protein and the targeting domain sequence, in order to facilitate selective binding to a particular cell type or tissue expressing a cell surface molecule that specifically binds to the polypeptide sequence ("targeting domain").

[0133] In some embodiments of the IL27Rα-binding molecule, the IL27Rα-binding molecule can be targeted to a specific cell type by incorporating a targeting domain into the structure of the IL27Rα-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 IL27Rα+ T cell.

[0134] 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.

[0135] In another aspect, the present disclosure provides a polyvalent binding molecule comprising (a) an IL27Rα binding molecule and (b) a second binding molecule that specifically binds to the extracellular domain of a second cell surface molecule, wherein the IL27Rα binding molecule and the second binding molecule are functionally linked, optionally via a chemical linker or polypeptide linker. In some embodiments, the IL27Rα 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 embodiments, 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).

[0136] In some embodiments, the targeting domain is a polypeptide selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Ra2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP, which specifically binds to cell surface molecules associated with tumor cells (e.g., cognitive ligands of tumor cell receptors).

[0137] In some embodiments, the targeting domain of the IL27Rα-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 IL27Rα-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.

[0138] 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.

[0139] sign In some embodiments, the IL27Rα-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 IL27Rα-binding sdAb (e.g., IL27Rα-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 an IL27Rα-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( 131I) 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.

[0140] Therapeutic drugs In some embodiments, the IL27Rα-binding molecule of this disclosure is functionally linked to a therapeutic agent. 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-derived molecules, or biological proteins (e.g., protein toxins) or particles (e.g., nanoparticles or recombinant viral particles, e.g., recombinant viral particles via viral coat proteins), therapeutic antibodies, and chemotherapeutic agents, as will be further fully described herein.

[0141] In some embodiments, therapeutic agents that may be incorporated into the IL27Rα-binding molecule of this disclosure are, for example, short-range radiation emitters including short-range, high-energy α- emitters. 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 213Bi), and Rhodium-188( 188 It contains Rh.

[0142] In some embodiments, the IL27Rα-binding molecule of the present disclosure is functionally linked to a cytotoxic agent (or its derivatives), such as mytansinol or DM1 mytansinoid, taxane, or caritiamicin, Pseudomonas exotoxin A, debuganin, lysine toxin, diphtheria toxin, amatoxin, for example, α-amanitin, saporin, mytansin, mytansinoid, auristatin, anthracycline, caritiamicin, irinotecan, SN-38, duocalmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer, or a variant thereof.

[0143] Synthesis of IL27Rα-binding molecules: In some embodiments, the IL27Rα-binding molecule of this disclosure is a polypeptide. However, in some embodiments, only a portion of the IL27Rα-binding molecule is a polypeptide, for example, the IL27Rα-binding molecule includes a non-peptidyl domain (e.g., PEG IL27Rα-binding sdAb conjugate, radionucleotide IL27Rα-binding sdAb conjugate, or small molecule IL27Rα-binding sdAb conjugate). The following provides guidance to enable solid-phase and recombinant synthesis of the polypeptide portion (domain) of the IL27Rα-binding molecule of this disclosure. In embodiments where only a portion of the IL27Rα-binding molecule is a polypeptide, the peptidyl domain of the IL27Rα-binding molecule will be understood as a process intermediate that may undergo further processing to complete the synthesis of the desired IL27Rα-binding molecule. The polypeptide domain of the IL27Rα-binding molecule may be produced by conventional methodologies for polypeptide construction, including recombinant or solid-phase synthesis, as described in more detail below.

[0144] 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 IL27Rα-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 IL27Rα-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).

[0145] In some embodiments, the polypeptide domain of the IL27Rα-binding molecule of this disclosure can be produced by chemical synthesis. The chemical synthesis of the polypeptide domain of the IL27Rα-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 IL27Rα-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, the α-functional group and any reactive side chain may be protected by an acid-unstable or basic-acid-unstable group that is stable under conditions for amide bond linking but can be easily cleaved without damaging the formed peptide chain.

[0146] 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.

[0147] 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 various chromatographic methods, including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high-pressure liquid chromatography (HPLC), and reversed-phase HPLC.

[0148] Recombinant production Alternatively, the polypeptide domain of the IL27Rα-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 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.

[0149] Synthesis of nucleic acid sequences encoding IL27Rα-binding molecules In some embodiments, the polypeptide domain of the IL27Rα-binding molecule is produced by a recombinant method using a nucleic acid sequence encoding the polypeptide domain of the IL27Rα-binding molecule (or a fusion protein containing the polypeptide domain of the IL27Rα-binding molecule). The nucleic acid sequence encoding the desired polypeptide domain of the IL27Rα-binding molecule can be synthesized by chemical means using an oligonucleotide synthesizer.

[0150] 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 IL27Rα-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.

[0151] The nucleic acid molecules encoding the polypeptide domain (and its fusion) of the IL27Rα-binding molecule may contain a natural 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).

[0152] The nucleic acid sequences encoding the polypeptide domain of the IL27Rα-binding molecule may be obtained from various commercial suppliers that provide custom synthesis of nucleic acid sequences. The amino acid sequence variants of the human IL27Rα-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.

[0153] Methods for constructing DNA sequences encoding the polypeptide domain of the IL27Rα-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 IL27Rα-binding molecule can also be introduced using standard recombination methods. In the case of deletions or additions, optionally digest the nucleic acid molecule encoding the polypeptide domain of the IL27Rα-binding molecule with an appropriate 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.

[0154] The polypeptide domain of the IL27Rα-binding molecule of this disclosure may be produced not only directly by recombination, but also as a fusion polypeptide with heterologous polypeptides, such as a signal sequence, or other polypeptides having a specific cleavage site at the N-terminus or C-terminus of the mature IL27Rα-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 recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In some embodiments, the signal sequence is a signal sequence naturally associated with the IL27Rα-binding molecule (i.e., a human IL27Rα signal sequence). The incorporation of the signal sequence depends on whether it is desirable to secrete the IL27Rα-binding molecule from the recombinant cell from which the IL27Rα-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 the IL27Rα-binding molecule to be secreted extracellularly into the culture medium, as described in Singh, U.S. Patent No. 7,198,919B1.

[0155] If the polypeptide domain of the IL27Rα-binding molecule to be expressed is to be expressed as a chimeric protein (e.g., a fusion protein containing the IL27Rα-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 IL27Rα-binding molecule and a second sequence encoding all or part of the heterologous polypeptide. For example, the polypeptide domain of the IL27Rα-binding molecule described herein may be fused with a hexahistidine tag (SEQ ID NO: 195) to facilitate the purification of the protein expressed by bacteria, or with a hexahistidine tag (SEQ ID NO: 195), hemagglutinin, or Fc 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 IL27Rα-binding molecule. For example, the N-terminus may be linked to a targeting domain, and the C-terminus may be linked to a hexahistidine tag (SEQ ID NO:195) purification handle.

[0156] A back-translated gene can be constructed using the complete amino acid sequence of the polypeptide domain of the IL27Rα-binding molecule (or fusion / chimera) to be expressed. DNA oligomers containing the nucleotide sequence encoding the polypeptide domain of the IL27Rα-binding molecule can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated together. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0157] In some embodiments, the nucleic acid sequence encoding the polypeptide domain of the IL27Rα-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 online tools exist to provide 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.

[0158] Expression vector Once assembled (by synthesis, site-directed mutagenesis, or other means), the nucleic acid sequence encoding the polypeptide domain of the IL27Rα-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.

[0159] 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.

[0160] The expression vectors for the polypeptide domain of the IL27Rα-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 IL27Rα-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 IL27Rα binding molecule, with respect to its potential secondary structure.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] host cell Furthermore, this disclosure provides prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding the polypeptide domain of an IL27Rα-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 IL27Rα-binding molecule, has been introduced by recombinant DNA. Progeny of such cells are also considered to be within the scope of this disclosure.

[0167] 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 IL27Rα-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.

[0168] In some embodiments, recombinant polypeptide domains of IL27Rα-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)).

[0169] 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.

[0170] The polypeptide domain of the IL27Rα-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).

[0171] In some embodiments, the recombinant polypeptide domain of the IL27Rα-binding molecule may or may not be glycosylated, depending on the host organism used to produce the IL27Rα-binding molecule. When bacteria are selected as the host, the polypeptide domain of the IL27Rα-binding sdAb may contain a glycosylation motif, in particular an N-linked glycosylation motif of the sequence Asn-X-Ser(NXS) or Asn-X-Thr(NXT), where X is any amino acid except proline. In such cases, it is desirable to eliminate the N-linked glycosylation motif by modifying the sequence of such an N-linked glycosylation motif to inhibit glycosylation. In some embodiments, the elimination of the Asn-X-Ser(NXS)N-linked glycosylation motif can be achieved by incorporating a conservation amino acid substitution of the Asn(N) and / or Ser(S) residues 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 can 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, when producing recombinant Criss recombinant IL27Rα-linked sdAb using a prokaryotic expression system, the prokaryotic host cell does not provide a glycosylation mechanism for the recombinant protein, so sequence modifications to exclude the N-linked glycosylation site may not be necessary.

[0172] 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.).

[0173] Transfection The expression construct can be introduced into host cells to produce the recombinant polypeptide domain of the IL27Rα-binding molecule disclosed herein, or to produce 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.

[0174] 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).

[0175] 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.

[0176] Recombinant protein recovery If a secretion leader sequence is used, the IL27Rα-binding polypeptide produced by recombination can be recovered from the culture medium as a secretion polypeptide. Alternatively, the IL27Rα-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.

[0177] purification Various purification processes are known and used in the art, such as affinity chromatography. 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, by 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.

[0178] The recombinant polypeptide domain of the IL27Rα-binding molecule produced by the transformed host can be purified according to any suitable method. The IL27Rα-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 IL27Rα-binding molecule.

[0179] Recombinant polypeptides in substantially purified form can be used as therapeutic agents, for example, as described herein.

[0180] The biological activity of the recombinant polypeptide domain of the IL27Rα-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 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA). This can be confirmed by IL27Rα 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).

[0181] How to use Inhibition of IL27Rα activity In one embodiment, the Disclosure provides a method for modulating the activity of IL27Rα-expressing cells by targeting and administering an IL27Rα-binding molecule in an amount sufficient to interfere with the activity of an IL27Rα-containing receptor. Furthermore, the Disclosure provides a method for modulating the activity of IL27Rα-expressing cells in a mixed cell population, comprising the step of contacting the cell population with the IL27Rα-binding molecule or complex of the Disclosure in vivo and / or ex vivo in an amount sufficient to interfere with the activity of an IL27Rα-containing receptor.

[0182] Identification, isolation, enrichment, or depletion of IL27Rα+ cells In one embodiment, the Disclosure provides a method for using the IL27Rα-binding molecule of the Disclosure useful in a process for isolating, concentrating, or depleting IL27Rα+ cells from a biological sample containing IL27Rα+ cells. The biological sample may include T cells, B cells, etc., of blood origin, e.g., PBMCs, or of cell culture origin or tissue origin such as brain or bone marrow. Processes suitable for isolating, concentrating, or depleting IL27Rα+ cells include centrifugation, filtration, magnetic cell sorting, and fluorescent cell sorting by techniques well known in the Art. Furthermore, the Disclosure provides a method for treating subjects suffering from a disease, disorder, or condition by administering a therapeutically effective amount of cell product obtained by concentrating or depleting IL27Rα+ cells using the IL27Rα-binding molecule as described herein.

[0183] In one embodiment, an IL27Rα-binding molecule containing a fluorescent label is used in the sorting procedure for use in FACS isolation or depletion of IL27Rα+ cells from a sample. The fluorescent label may be attached directly to the sdAb of the IL27Rα-binding molecule (e.g., optionally by chemical conjugation using a linker) or indirectly (e.g., by biotinylation of the sdAb and binding of a biotinylated antibody to a streptavidin fluorescent dye conjugate). Such fluorescently labeled IL27Rα+ cells can be isolated from a mixed cell population using conventional FACS techniques.

[0184] In another embodiment, the selection procedure uses an IL27Rα-binding molecule of the Disclosure (e.g., IL27Rα-binding VHH) conjugated to magnetic particles that result in the magnetic labeling of IL27Rα+ cells for use in a magnetic cell separation procedure. In one embodiment, the method includes (a) conjugating one or more IL27Rα-binding molecules of the Disclosure (e.g., IL27Rα-binding VHH) to magnetic particles; (b) preparing a mixture by contacting a biological sample with a certain amount of magnetic particles conjugated to an IL27Rα-binding molecule; (c) subjecting the mixture to a magnetic field so that magnetically labeled IL27Rα+ cells are retained; (d) removing unlabeled cells from the mixture; and (e) removing the magnetic field to isolate the IL27Rα+ cells.

[0185] Cell selection procedures (e.g., FACS or magnetic separation) yield two products: (a) a cell population depleted of IL27Rα+ cells and (b) a cell population enriched with IL27Rα+ cells. Each of these populations can be further processed by conventional procedures to identify specific IL27Rα+ cell subsets or IL27Rα- cell subsets that may be useful for research, diagnostic, or clinical applications. For example, isolation of specific IL27Rα+ T cell subsets that also express one or more of CD4, CD8, CD19, CD25, and CD62L by FACS or magnetic separation using techniques well known in the art, followed by further iteration using one or more antibodies that specifically bind to the CD4, CD8, CD19, CD25, and CD62L antigens, respectively.

[0186] In one embodiment, the IL27Rα-binding molecule can be used to deplete IL27Rα-expressing cells from a biological sample containing IL27Rα-expressing cells, such as peripheral blood or lymphoid tissue, which may optionally be further processed for further isolation of an IL27Rα+ naive T cell subset, isolation of human IL27Rα+ memory T cells from a population of CD4+ or CD8+ cells, or isolation of human IL27RαRA+ naive T cells from pre-selected CD4+ or CD8+ cells by depletion of IL27Rα+ cells. In one embodiment, the IL27Rα-binding molecule provides a method for preparing a population of naive Treg-enriched cells from a biological sample, comprising the step of depleting IL27Rα+ cells using the IL27Rα-binding molecule of the Disclosure as described above, and optionally further comprising the step of depleting CD8+ and / or CD19+ cells. The IL27Rα+ depleted cell population may optionally be further expanded in vitro for specific cell types in the preparation of a cell product containing a therapeutically effective amount of IL27Rα+ depleted cell product that can be administered to subjects suffering from a disease, disorder, or condition. The IL27Rα+ enriched cell population may optionally be further expanded in vitro in the preparation of a cell product containing a therapeutically effective amount of IL27Rα+ cells.

[0187] kit This disclosure also intends to provide kits containing pharmaceutical compositions of IL27Rα-binding molecules. The kits generally take the form of physical structures containing various components, as described below, and can be used, for example, in the implementation of the methods described above. The kits of this disclosure can be designed for conditions necessary to properly maintain the components contained in the kit (e.g., freezing or refrigeration). The kits may further include labels or accompanying documents containing identifying information for 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 labels or accompanying documents may contain manufacturer information such as lot number and expiration date. The labels or accompanying documents may, for example, be 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 labels or accompanying documents may be provided in physical form or on computer-readable media. 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 through secure access by providing a password (or a scannable code such as a barcode or QR code located on the container of the IL27Rα-binding molecule or on the surface of the kit containing it), in accordance with government regulations (e.g., HIPAA). [Examples]

[0188] The following examples are provided to fully disclose and illustrate to those skilled in the art how to prepare and use the IL27Rα-binding molecule, and are not intended to limit the scope of what the inventors consider to be the IL27Rα-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 IL27Rα-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.

[0189] 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.

[0190] Example 1. Immunization Protocol The process for isolating anti-hIL27RαVHH was initiated by immunizing camels with polypeptides corresponding to amino acids 33-516 of hIL27Rα (UNIPROT reference number Q6UWB1). The process for isolating anti-mIL27RαVHH was initiated by immunizing camels with the 201 amino acid extracellular domain of mIL27Rα and amino acids 25-510 of the mIL27Rα precursor (UNIPROT reference number O70394). For each antigen, VHH was identified and isolated using the following methodology.

[0191] 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.

[0192] 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.

[0193] Example 3: Isolation of antigen-specific VHH Biopanning of a phage library was performed to identify VHH that binds to IL27Rα. A 96-well plate was coated with IL27Rα, and the phage library was incubated in each well so that phage-expressed IL27Rα-reactive VHH could bind to the IL27Rα on the plate. Nonspecifically bound phages were washed away, and specifically bound phages were isolated. After selection, the enriched phage library expressing IL27Rα-reactive VHH was amplified in TG1 cells. The biopanning process described above was repeated 2-3 times to enrich the library for IL27Rα-reactive VHH.

[0194] Example 4: Identification of antibodies that specifically bind to IFNgR1: 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 IL27Rα-coated plates to identify positive VHH conjugates selectively bound to IFNgR1. The 96-well plates were coated with IL27Rα 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 defined as specifically binding to IL27Rα. Positive clones were sequenced, and their sequences were analyzed to identify unique chronotypes.

[0195] Example 5. Evaluation of coupling affinity via surface plasmon resonance To evaluate binding via SPR as described below, representative examples were selected from each hIL27RαVHH chronotype prepared according to Examples 1-3. The binding affinity of hIL27Rα binding molecules corresponding to SEQ ID NOs 2-27 was evaluated using surface plasmon resonance (SPR) in general according to the following procedure. All experiments were performed in a Biacore T200 instrument equipped with a protein A derivatization sensor chip (Cytiva) in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20), and 3 mM EDTA (HBS-EP+ buffer). Mono-Fc VHH ligand was flowed at 5 μl / min for a variable time of 18-300 seconds until the capture load listed in the table below was reached. After ligand capture, a series of 2-fold dilutions of the extracellular domain of the IL27Rα receptor, modified to incorporate a C-terminal polyHis sequence, was injected in high-performance mode or single-cycle kinetics mode, typically containing at least five different concentrations ranging from 1 μM to 1 nM. Surface regeneration was performed by flowing 10 mM glycine-HCl, pH 1.5 (60 seconds, 50 μL / min). Sensograms with buffer subtracted were processed with Biacore T200 Evaluation Software to determine the rate constant and affinity constant (k a , k d , K D A 1:1 Langmuir-coupled model was used for global fitting to extract the relevant components (bulk shift set to 0). MAX <100RU indicates a surface density suitable for reaction rate analysis. max The calculated values ​​were prepared using the formula: Rmax = load (RU) x ligand binding valency x (molecular weight of analyte / molecular weight of ligand). Surface activity was defined as the ratio of the experimental Rmax value to the calculated Rmax value. The results of these binding affinity experiments are shown in Table 6.

[0196] 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.

[0197] Sequence information SEQUENCE LISTING <110> SYNTHEKINE, INC. <120> IL27RALPHA 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> 202 <170> PatentIn version 3.5 <210> 1 <211> 636 <212> PRT <213> Homo sapiens <400> 1 Met Arg Gly Gly Arg Gly Ala Pro Phe Trp Leu Trp Pro Leu Pro Lys 1 5 10 15 Leu Ala Leu Leu Pro Leu Leu Trp Val Leu Phe Gln Arg Thr Arg Pro 20 25 30 Gln Gly Ser Ala Gly Pro Leu Gln Cys Tyr Gly Val Gly Pro Leu Gly 35 40 45 Asp Leu Asn Cys Ser Trp Glu Pro Leu Gly Asp Leu Gly Ala Pro Ser 50 55 60 Glu Leu His Leu Gln Ser Gln Lys Tyr Arg Ser Asn Lys Thr Gln Thr 65 70 75 80 Val Ala Val Ala Ala Gly Arg Ser Trp Val Ala Ile Pro Arg Glu Gln 85 90 95 Leu Thr Met Ser Asp Lys Leu Leu Val Trp Gly Thr Lys Ala Gly Gln 100 105 110 Pro Leu Trp Pro Pro Val Phe Val Asn Leu Glu Thr Gln Met Lys Pro 115 120 125 Asn Ala Pro Arg Leu Gly Pro Asp Val Asp Phe Ser Glu Asp Asp Pro 130 135 140 Leu Glu Ala Thr Val His Trp Ala Pro Pro Thr Trp Pro Ser His Lys 145 150 155 160 Val Leu Ile Cys Gln Phe His Tyr Arg Arg Cys Gln Glu Ala Ala Trp 165 170 175 Thr Leu Leu Glu Pro Glu Leu Lys Thr Ile Pro Leu Thr Pro Val Glu 180 185 190 Ile Gln Asp Leu Glu Leu Ala Thr Gly Tyr Lys Val Tyr Gly Arg Cys 195 200 205 Arg Met Glu Lys Glu Glu Asp Leu Trp Gly Glu Trp Ser Pro Ile Leu 210 215 220 Ser Phe Gln Thr Pro Pro Ser Ala Pro Lys Asp Val Trp Val Ser Gly 225 230 235 240 Asn Leu Cys Gly Thr Pro Gly Gly Glu Glu Pro Leu Leu Leu Trp Lys 245 250 255 Ala Pro Gly Pro Cys Val Gln Val Ser Tyr Lys Val Trp Phe Trp Val 260 265 270 Gly Gly Arg Glu Leu Ser Pro Glu Gly Ile Thr Cys Cys Cys Ser Leu 275 280 285 Ile Pro Ser Gly Ala Glu Trp Ala Arg Val Ser Ala Val Asn Ala Thr 290 295 300 Ser Trp Glu Pro Leu Thr Asn Leu Ser Leu Val Cys Leu Asp Ser Ala 305 310 315 320 Ser Ala Pro Arg Ser Val Ala Val Ser Ser Ile Ala Gly Ser Thr Glu 325 330 335 Leu Leu Val Thr Trp Gln Pro Gly Pro Gly Glu Pro Leu Glu His Val 340 345 350 Val Asp Trp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val 355 360 365 Arg Leu Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr 370 375 380 Val Gly Val Pro Tyr Arg Ile Thr Val Thr Ala Val Ser Ala Ser Gly 385 390 395 400 Leu Ala Ser Ala Ser Ser Val Trp Gly Phe Arg Glu Glu Leu Ala Pro 405 410 415 Leu Val Gly Pro Thr Leu Trp Arg Leu Gln Asp Ala Pro Pro Gly Thr 420 425 430 Pro Ala Ile Ala Trp Gly Glu Val Pro Arg His Gln Leu Arg Gly His 435 440 445 Leu Thr His Tyr Thr Leu Cys Ala Gln Ser Gly Thr Ser Pro Ser Val 450 455 460 Cys Met Asn Val Ser Gly Asn Thr Gln Ser Val Thr Leu Pro Asp Leu 465 470 475 480 Pro Trp Gly Pro Cys Glu Leu Trp Val Thr Ala Ser Thr Ile Ala Gly 485 490 495 Gln Gly Pro Pro Gly Pro Ile Leu Arg Leu His Leu Pro Asp Asn Thr 500 505 510 Leu Arg Trp Lys Val Leu Pro Gly Ile Leu Phe Leu Trp Gly Leu Phe 515 520 525 Leu Leu Gly Cys Gly Leu Ser Leu Ala Thr Ser Gly Arg Cys Tyr His 530 535 540 Leu Arg His Lys Val Leu Pro Arg Trp Val Trp Glu Lys Val Pro Asp 545 550 555 560 Pro Ala Asn Ser Ser Ser Gly Gln Pro His Met Glu Gln Val Pro Glu 565 570 575 Ala Gln Pro Leu Gly Asp Leu Pro Ile Leu Glu Val Glu Glu Met Glu 580 585 590 Pro Pro Pro Val Met Glu Ser Ser Gln Pro Ala Gln Ala Thr Ala Pro 595 600 605 Leu Asp Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu 610 615 620 Gly Leu Leu Gly Pro Pro Arg Pro Gln Val Leu Ala 625 630 635 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 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 Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Thr Ile Ser Ala Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Ala Ala Ile Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Arg Arg Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 3 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 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 Phe Thr Phe Ser Leu Ser 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 100 105 110 Gly Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 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 Val Ala Ser Gly Tyr Val Ser Cys Asp Tyr 20 25 30 Phe Leu Pro Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ser Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Glu Asp Lys Gly Lys Asn Ile 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 Lys Ala Ser Cys Val Arg Gly Arg Ala Val Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 5 <211> 124 <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 Leu Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Val Ala Tyr Gly Ile Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Val Lys His Ser Gly Thr Thr Ile Pro Arg Gly Phe Ile Ser Tyr Thr 100 105 110 Lys Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 6 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 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 Ala Ser Gly Tyr Val Ser Cys Asp Tyr 20 25 30 Phe Leu Pro Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ser Val Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Gln Asp Lys Gly Lys Asn Ile 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 Lys Ala Ser Cys Val Arg Gly Arg Ala Ile Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 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 Phe Ser Phe Ser Ser Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Ser Gly Gly Ser Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Ile Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Ala Ile Val Pro Thr Gly Ala Thr Met Glu Arg Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 8 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 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 Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Thr Ile Ser Ala Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Arg Arg Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 9 <211> 123 <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 Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Thr Ile Ser Ala Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Arg Arg Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 10 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Ser Thr Ser 20 25 30 Asn Ser Trp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ala Ala Ile Tyr Thr Val Gly Gly Ser Ile Phe Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Gln Asp Ala Thr Lys Asn Met 65 70 75 80 Phe Tyr Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Ala Ala Ser Gly Arg Leu Arg Gly Lys Trp Phe Trp Pro 100 105 110 Tyr Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 11 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 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 Arg Ala Ser Gly Ser Thr Tyr Ser Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ile Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Gln Ala Lys Asn Thr Leu 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 Ala Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Asp Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 12 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 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 Arg Ala Ser Gly Ser Thr Tyr Ser Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ser Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp His Ala Lys Asn Thr Val Thr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Gly Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 13 <211> 129 <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 Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Ser Thr Tyr Ser Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ile Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Gln Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Asp Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 14 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 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 Val Ala Ser Gly Tyr Val Ser Cys Asp Tyr 20 25 30 Phe Leu Pro Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ser Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Gln Asp Arg Gly Lys Asn Ile 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 Lys Ala Ser Cys Val Arg Gly Arg Thr Ile Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 15 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 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 Val Ala Ser Gly Tyr Val Ser Cys Asp Tyr 20 25 30 Phe Leu Pro Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ser Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Gln Asp Lys Gly Lys Asn Ile 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 Lys Ala Ser Cys Val Arg Gly Arg Ala Ile Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 16 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 16 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 Val Ala Ser Gly Tyr Val Ser Cys Asp Tyr 20 25 30 Phe Leu Pro Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ser Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Gln Asp Lys Gly Lys Asn Ile Ala Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Lys Ala Ser Cys Val Arg Gly Arg Ala Ile Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 17 <211> 129 <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 Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Ser Thr Tyr Ser Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ile Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Gln Ala Lys Asn Thr Val 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 Ala Glu Ser Ala Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Asp Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 18 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 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 Phe Thr Phe Ser Leu Ser 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 100 105 110 Gly Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 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 Val Ala Ser Gly Tyr Val Ser Cys Asp Tyr 20 25 30 Phe Leu Pro Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ser Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Gln Asp Lys Gly Lys Asn Ile 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 Lys Ala Ser Cys Val Arg Gly Arg Gly Ile Ser Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 20 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 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 Arg Ala Ser Gly Ser Thr Tyr Ser Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ile Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Gln Ala Lys Asn Thr Val 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 Ala Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Asp Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 21 <211> 123 <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 Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Ser Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Lys Arg Ser Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 22 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 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 Phe Thr Phe Ser Leu Ser 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 100 105 110 Gly Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 23 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 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 Arg Ala Ser Gly Ser Thr Tyr Ser Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Thr Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Gln Ala Lys Asn Thr Val 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 Ala Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Asp Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 24 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 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 Arg Ala Ser Arg Ser Pro Tyr Gly Asn Tyr 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ser Thr Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp His Ala Lys Asn Thr Val Thr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg 100 105 110 Pro Asp Arg Val Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 25 <211> 123 <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 Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Ser 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 100 105 110 Gly Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Phe Thr Phe Ser Ser Tyr Pro Met Ser 1 5 <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Thr Ile Ser Ala Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 28 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Arg Arg Asn Tyr 1 5 10 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Phe Thr Phe Ser Leu Ser Gly Met Ser 1 5 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Ala Ile Ser Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 31 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 1 5 10 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Tyr Val Ser Cys Asp Tyr Phe Leu Pro Ser 1 5 10 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val Lys Gly 1 5 10 15 <210> 34 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Ser Cys Val Arg Gly Arg Ala Val Ser Glu Tyr 1 5 10 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Phe Thr Phe Ser Asn Tyr Ala Met Ser 1 5 <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Gly Ile Asn Val Ala Tyr Gly Ile Thr Ser Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 His Ser Gly Thr Thr Ile Pro Arg Gly Phe Ile Ser Tyr Thr Lys 1 5 10 15 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 Tyr Val Ser Cys Asp Tyr Phe Leu Pro Ser 1 5 10 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Val Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val Lys Gly 1 5 10 15 <210> 40 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Ser Cys Val Arg Gly Arg Ala Ile Ser Glu Tyr 1 5 10 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Phe Ser Phe Ser Ser Tyr Ala Met Lys 1 5 <210> 42 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Thr Ile Ser Ser Gly Gly Ser Ser Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Ala Ile Val Pro Thr Gly Ala Thr Met Glu 1 5 10 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Phe Thr Phe Ser Ser Tyr Pro Met Ser 1 5 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Thr Ile Ser Ala Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Arg Arg Asn Tyr 1 5 10 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Phe Thr Phe Ser Ser Tyr Pro Met Ser 1 5 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Thr Ile Ser Ala Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 49 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 49 Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Arg Arg Asn Tyr 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Phe Thr Tyr Ser Thr Ser Asn Ser Trp Met Ala 1 5 10 <210> 51 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 Ala Ile Tyr Thr Val Gly Gly Ser Ile Phe Tyr Ala Asp Ser Val Arg 1 5 10 15 Gly <210> 52 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Ala Ser Gly Arg Leu Arg Gly Lys Trp Phe Trp Pro Tyr Glu Tyr Asn 1 5 10 15 Tyr <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Ser Thr Tyr Ser Asn Tyr Cys Leu Gly 1 5 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 55 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Asp 1 5 10 15 Arg Val Pro Tyr 20 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Ser Thr Tyr Ser Asn Tyr Cys Leu Gly 1 5 <210> 57 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 58 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Gly 1 5 10 15 Arg Val Pro Tyr 20 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Ser Thr Tyr Ser Asn Tyr Cys Leu Gly 1 5 <210> 60 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 61 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Asp 1 5 10 15 Arg Val Pro Tyr 20 <210> 62 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Tyr Val Ser Cys Asp Tyr Phe Leu Pro Ser 1 5 10 <210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val Lys Gly 1 5 10 15 <210> 64 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Ser Cys Val Arg Gly Arg Thr Ile Ser Glu Tyr 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Tyr Val Ser Cys Asp Tyr Phe Leu Pro Ser 1 5 10 <210> 66 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val Lys Gly 1 5 10 15 <210> 67 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Ser Cys Val Arg Gly Arg Ala Ile Ser Glu Tyr 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Tyr Val Ser Cys Asp Tyr Phe Leu Pro Ser 1 5 10 <210> 69 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 69 Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val Lys Gly 1 5 10 15 <210> 70 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Ser Cys Val Arg Gly Arg Ala Ile Ser Glu Tyr 1 5 10 <210> 71 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Ser Thr Tyr Ser Asn Tyr Cys Leu Gly 1 5 <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 72 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 73 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 73 Glu Ser Ala Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Asp 1 5 10 15 Arg Val Pro Tyr 20 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 74 Phe Thr Phe Ser Leu Ser Gly Met Ser 1 5 <210> 75 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 Ala Ile Ser Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 76 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 76 Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 1 5 10 <210> 77 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 77 Tyr Val Ser Cys Asp Tyr Phe Leu Pro Ser 1 5 10 <210> 78 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Ile Ile Asp Gly Thr Gly Ser Thr Ser Tyr Ala Ala Ser Val Lys Gly 1 5 10 15 <210> 79 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Ser Cys Val Arg Gly Arg Gly Ile Ser Glu Tyr 1 5 10 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Ser Thr Tyr Ser Asn Tyr Cys Leu Gly 1 5 <210> 81 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 81 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 82 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 82 Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Asp 1 5 10 15 Arg Val Pro Tyr 20 <210> 83 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Phe Thr Phe Ser Ser Tyr Pro Met Ser 1 5 <210> 84 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Thr Ile Ser Ser Gly Gly Asp Thr Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 85 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Arg Ile Asp Cys Asn Ser Gly Tyr Cys Tyr Lys Arg Ser Tyr 1 5 10 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Phe Thr Phe Ser Leu Ser Ser Met Ser 1 5 <210> 87 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 Ala Ile Ser Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 88 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 1 5 10 <210> 89 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Ser Thr Tyr Ser Asn Tyr Cys Leu Gly 1 5 <210> 90 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 91 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Asp 1 5 10 15 Arg Val Pro Tyr 20 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Ser Pro Tyr Gly Asn Tyr Cys Leu Gly 1 5 <210> 93 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Val Ile Asn Trp Val Gly Gly Met Leu Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 94 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 94 Glu Ser Val Ser Ser Phe Ser Cys Gly Gly Trp Leu Thr Arg Pro Asp 1 5 10 15 Arg Val Pro Tyr 20 <210> 95 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 95 Phe Thr Phe Ser His Ser Gly Met Ser 1 5 <210> 96 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 96 Thr Ile Asn Ser Gly Gly Ala Ser Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 97 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 97 Gly Gly Ser Gly Tyr Gly Asp Ala Ser Arg Met Thr Ser Pro 1 5 10 <210> 98 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 98 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacccca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg gatcagcacc atcagcgccg gcggcgacac caccctgtac 180 gccgacagcg tgaagggcag gttcaccagc agcagggaca acgccaagaa caccctgtac 240 ctgcagctga acagcctgaa gaccgaggac gccgccatct actactgcgc caagaggatc 300 gactgcaaca gcggctactg ctacaggagg aactactggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 99 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 99 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc ctgagcggca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcgcc atcagcagcg gcggcgccag cacctactac 180 accgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa catcctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatgt actactgcgc caagggcggc 300 agcggctacg gcgacgccag caggatgacc agccccggca gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 100 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 100 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgtgg ccagcggcta cgtgagctgc gactacttcc tgcccagctg gtacaggcag 120 gcccccggca aggagaggga gttcgtgagc atcatcgacg gcaccggcag caccagctac 180 gccgccagcg tgaagggcag gttcaccgcc agcgaggaca agggcaagaa catcgcctac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcaa ggccagctgc 300 gtgaggggca gggccgtgag cgagtactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 101 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 101 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcgagag cctgaggctg 60 agctgcaccg ccagcggctt caccttcagc aactacgcca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcggc atcaacgtgg cctacggcat caccagctac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acaccaagaa caccctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatct actactgcgt gaagcacagc 300 ggcaccacca tccccagggg cttcatcagc tacaccaaga ggggccaggg cacccaggtg 360 accgtgagca gc 372 <210> 102 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 102 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaccg ccagcggcta cgtgagctgc gactacttcc tgcccagctg gtacaggcag 120 gcccccggca aggagaggga gttcgtgagc gtgatcgacg gcaccggcag caccagctac 180 gccgccagcg tgaagggcag gttcaccgcc agccaggaca agggcaagaa catcgcctac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcaa ggccagctgc 300 gtgaggggca gggccatcag cgagtactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 103 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 103 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt cagcttcagc agctacgcca tgaagtgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcacc atcagcagcg gcggcagcag caccaactac 180 gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccctgtac 240 ctgcagctga acagcctgaa gatcgaggac accgccatgt actactgcgc caaggccatc 300 gtgcccaccg gcgccaccat ggagaggggc cagggcaccc aggtgaccgt gagcagc 357 <210> 104 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 104 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacccca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg gatcagcacc atcagcgccg gcggcgacac caccctgtac 180 gccgacagcg tgaagggcag gttcaccagc agcagggaca acgccaagaa caccctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatct actactgcgc caagaggatc 300 gactgcaaca gcggctactg ctacaggagg aactactggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 105 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 105 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg tgggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacccca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg gatcagcacc atcagcgccg gcggcgacac caccctgtac 180 gccgacagcg tgaagggcag gttcaccagc agcagggaca acgccaagaa caccctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatct actactgcgc caagaggatc 300 gactgcaaca gcggctactg ctacaggagg aactactggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 106 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 106 caggtgcagc tgcaggagag cggcggcggc agcgtgcaga gcggcggcag cctgaggctg 60 agctgcgccg ccagcggctt cacctacagc accagcaaca gctggatggc ctggttcagg 120 caggcccccg gcaaggagag ggagggcgtg gccgccatct acaccgtggg cggcagcatc 180 ttctacgccg acagcgtgag gggcaggttc accatcagcc aggacgccac caagaacatg 240 ttctacctgc agatgaacac cctgaagccc gaggacaccg ccatgtacta ctgcgccgcc 300 gccagcggca ggctgagggg caagtggttc tggccctacg agtacaacta ctggggccag 360 ggcacccagg tgaccgtgag cagc 384 <210> 107 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 107 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaggg ccagcggcag cacctacagc aactactgcc tgggctggtt caggcagatc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc aggccaagaa caccctgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgagagc 300 gtgagcagct tcagctgcgg cggctggctg accaggcccg acagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 108 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 108 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaggg ccagcggcag cacctacagc aactactgcc tgggctggtt caggcagagc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc acgccaagaa caccgtgacc 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgagagc 300 gtgagcagct tcagctgcgg cggctggctg accaggcccg gcagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 109 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 109 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcgagag cctgaggctg 60 agctgcaggg ccagcggcag cacctacagc aactactgcc tgggctggtt caggcagatc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc aggccaagaa caccgtgtac 240 ctggagatga acagcctgaa gcccgaggac accgccatgt actactgcgc caccgagagc 300 gtgagcagct tcagctgcgg cggctggctg accaggcccg acagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 110 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 110 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgtgg ccagcggcta cgtgagctgc gactacttcc tgcccagctg gtacaggcag 120 gcccccggca aggagaggga gttcgtgagc atcatcgacg gcaccggcag caccagctac 180 gccgccagcg tgaagggcag gttcaccgcc agccaggaca ggggcaagaa catcgcctac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcaa ggccagctgc 300 gtgaggggca ggaccatcag cgagtactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 111 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 111 60. caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg agctgcgtgg ccagcggcta cgtgagctgc gactacttcc tgcccagctg gtacaggcag gcccccggca aggagagga gttcgtgagc atcatcgacg gcaccggcag caccagctac 180 gccgccagcg tgaagggcag gttcaccgcc agccaggaca agggcaaga catcgcctac 240 300. ctgcagatga acagcctga gcccgaggac accgccatgt actactgcaa ggccagctgc gtgaggggca gggccatcag cgagtactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 112 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 112 60. caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg agctgcgtgg ccagcggcta cgtgagctgc gactacttcc tgcccagctg gtacaggcag gcccccggca aggagagga gttcgtgagc atcatcgacg gcaccggcag caccagctac 180 gccgccagcg tgaagggcag gttcaccgcc agccaggaca agggcaagaa catcgcctac 240 ctgcagatga acaccctgaa gcccgaggac accgccatgt actactgcaa ggccagctgc 300 gtgaggggca gggccatcag cgagtactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 113 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 113 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaggg ccagcggcag cacctacagc aactactgcc tgggctggtt caggcagatc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc aggccaagaa caccgtgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgagagc 300 gccagcagct tcagctgcgg cggctggctg accaggcccg acagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 114 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 114 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc ctgagcggca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcgcc atcagcagcg gcggcgccag cacctactac 180 accgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa catgctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatgt actactgcgc caagggcggc 300 agcggctacg gcgacgccag caggatgacc agccccggca gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 115 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 115 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgtgg ccagcggcta cgtgagctgc gactacttcc tgcccagctg gtacaggcag 120 gcccccggca aggagaggga gttcgtgagc atcatcgacg gcaccggcag caccagctac 180 gccgccagcg tgaagggcag gttcaccgcc agccaggaca agggcaagaa catcgcctac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcaa ggccagctgc 300 gtgaggggca ggggcatcag cgagtactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 116 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 116 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaggg ccagcggcag cacctacagc aactactgcc tgggctggtt caggcagatc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc aggccaagaa caccgtgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgagagc 300 gtgagcagct tcagctgcgg cggctggctg accaggcccg acagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 117 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 117 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacccca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcacc atcagcagcg gcggcgacac caccctgtac 180 gccgacagcg tgaagggcag gttcaccagc agcagggaca acgccaagaa caccctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatgt actactgcgc caagaggatc 300 gactgcaaca gcggctactg ctacaagagg agctactggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 118 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 118 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc ctgagcagca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgagcgcc atcagcagcg gcggcgccag cacctactac 180 accgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa catgctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatgt actactgcgc caagggcggc 300 agcggctacg gcgacgccag caggatgacc agccccggca gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 119 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 119 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaggg ccagcggcag cacctacagc aactactgcc tgggctggtt caggcagacc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc aggccaagaa caccgtgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgagagc 300 gtgagcagct tcagctgcgg cggctggctg accaggcccg acagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 120 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 120 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaggg ccagcaggag cccctacggc aactactgcc tgggctggtt caggcagagc 120 accggcaagg agagggaggg cgtggccgtg atcaactggg tgggcggcat gctgtacttc 180 gccgacagcg tgaagggcag gttcaccgtg agccaggacc acgccaagaa caccgtgacc 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgagagc 300 gtgagcagct tcagctgcgg cggctggctg accaggcccg acagggtgcc ctactggggc 360 cagggcaccc aggtgaccgt gagcagc 387 <210> 121 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 121 caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc cacagcggca tgagctgggt gaggcaggcc 120 cccggcaagg gcctggagtg gtgagcacc atcacagcg gcggcgccag cacctactac 180 accgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa catgctgtac 240 ctgcagctga acagcctgaa gaccgaggac accgccatgt actactgcgc caagggcggc 300 agcggctacg gcgacgccag caggatgacc agccccggca gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 122 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 122 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 Lys Asn Ser Asn Phe Met Gly 20 25 30 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Met 35 40 45 Met Thr Lys Asn Asn Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser His Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met 65 70 75 80 Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val 85 90 95 Tyr Arg Thr Arg Arg Leu Arg Val Leu Glu Ala Ala Asn Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 123 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 123 Asn Ser Asn Phe Met Gly 1 5 <210> 124 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 124 Ala Met Met Thr Lys Asn Asn Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 125 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 125 Val Tyr Arg Thr Arg Arg Leu Arg Val Leu Glu Ala Ala Asn Phe Asp 1 5 10 15 Tyr <210> 126 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 126 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 Ala Ser Gly Tyr Thr Ser Ser Arg Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Lys Arg Glu Gly Val 35 40 45 Ala Ala Ile Tyr Thr Gly Gly Gly Thr Thr Phe Tyr His Gly Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Thr Thr Asn Thr Val Tyr 65 70 75 80 Leu Gln Met His Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Gly Pro Val Thr Arg Ala Cys Asp Glu Tyr Asn Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 127 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 127 Tyr Thr Ser Ser Arg Tyr Cys Met Gly 1 5 <210> 128 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 128 Ala Ile Tyr Thr Gly Gly Gly Thr Thr Phe Tyr His Gly Ser Val Lys 1 5 10 15 Gly <210> 129 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 129 Gly Pro Val Thr Arg Ala Cys Asp Glu Tyr Asn Tyr 1 5 10 <210> 130 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 130 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 Gly Ser Gly Tyr Ser Leu Ser Asn Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Arg Glu Gly Val 35 40 45 Ala Ser Leu Arg Phe Val Ser Gly Ala Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ala Gln Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Gly Ile Lys Ser Arg Gly Ile Cys Gly Gly Arg Leu Val Asp Val Asp 100 105 110 Phe Gly Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 131 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 131 Tyr Ser Leu Ser Asn Tyr Cys Met Gly 1 5 <210> 132 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 132 Ser Leu Arg Phe Val Ser Gly Ala Thr Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 133 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 133 Lys Ser Arg Gly Ile Cys Gly Gly Arg Leu Val Asp Val Asp Phe Gly 1 5 10 15 Asn <210> 134 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 134 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 Ile Asn Arg Met 20 25 30 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Ser Ile Gly Gly Gly Gln Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Asp Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala 85 90 95 Gly Leu Val Tyr Gly Glu Ala Trp Leu Asp Ser Arg His Tyr Asn Lys 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 135 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 135 Tyr Ser Ile Asn Arg Met Gly 1 5 <210> 136 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 136 Ala Ile Ser Ile Gly Gly Gly Gln Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 137 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 137 Gly Leu Val Tyr Gly Glu Ala Trp Leu Asp Ser Arg His Tyr Asn Lys 1 5 10 15 <210> 138 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 138 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Ala Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His 85 90 95 Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 139 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 139 Asp Ser Thr Tyr Ser Met Gly 1 5 <210> 140 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 140 Ala Ile Ala Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 141 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 141 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 142 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 142 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 Ala 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 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 Ala Ser Gly Arg Cys Leu Gly Pro Gly Ile Arg Ser Leu Ile Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 143 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 143 Tyr Thr Tyr Ser Ser Tyr Cys Met Ala 1 5 <210> 144 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 144 Ala Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 145 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 145 Ala Ser Gly Arg Cys Leu Gly Pro Gly Ile Arg Ser Leu Ile 1 5 10 <210> 146 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 146 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Thr Lys Asp Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn 65 70 75 80 Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His Arg 85 90 95 Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 147 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 147 Asp Ser Thr Tyr Ser Met Gly 1 5 <210> 148 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 148 Ala Ile Thr Lys Asp Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 149 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 149 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 150 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 150 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Pro Thr Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His 85 90 95 Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 151 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 151 Asp Ser Thr Tyr Ser Met Gly 1 5 <210> 152 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 152 Ala Ile Pro Thr Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 153 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 153 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 154 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 154 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Ala Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His 85 90 95 Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 155 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 155 Asp Ser Thr Tyr Ser Met Gly 1 5 <210> 156 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 156 Ala Ile Ala Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 157 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 157 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 158 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 158 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Gly Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His 85 90 95 Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 159 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 159 Asp Ser Thr Tyr Ser Met Gly 1 5 <210> 160 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 160 Ala Ile Gly Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 161 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 161 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 162 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 162 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Thr Lys Asp Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn 65 70 75 80 Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His Arg 85 90 95 Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 163 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 163 Asp Ser Thr Tyr Ser Met Gly 1 5 <210> 164 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 164 Ala Ile Thr Lys Asp Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 165 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 165 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 166 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 166 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 Ser Ile Asn Arg Met 20 25 30 Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Ser Ile Gly Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys His Thr Val Asp Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala 85 90 95 Gly Leu Val Tyr Gly Glu Ala Trp Leu Asp Ser Arg His Tyr Asn Lys 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 167 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 167 Tyr Ser Ile Asn Arg Met Ala 1 5 <210> 168 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 168 Ala Ile Ser Ile Gly Gly Asp Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 169 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 169 Gly Leu Val Tyr Gly Glu Ala Trp Leu Asp Ser Arg His Tyr Asn Lys 1 5 10 15 <210> 170 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 170 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 Ile Asn Arg Met 20 25 30 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Ser Ile Gly Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Asp Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala 85 90 95 Gly Leu Val Tyr Gly Glu Ala Trp Leu Asp Ser Arg His Tyr Asn Lys 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 171 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 171 Tyr Ser Ile Asn Arg Met Gly 1 5 <210> 172 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 172 Ala Ile Ser Ile Gly Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 173 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 173 Gly Leu Val Tyr Gly Glu Ala Trp Leu Asp Ser Arg His Tyr Asn Lys 1 5 10 15 <210> 174 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 174 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 Val Ser Gly Asp Ser Thr Tyr Ser Met 20 25 30 Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Thr Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly Arg 50 55 60 Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala His 85 90 95 Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 175 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 175 Asp Ser Thr Tyr Ser Met 1 5 <210> 176 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 176 Ala Ile Thr Lys Asp Gly Ile Thr Ile His Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 177 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 177 His Arg Pro Tyr Gly Pro Pro Leu Asn Pro Arg Trp Tyr Thr Tyr 1 5 10 15 <210> 178 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 178 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg ccagcaagaa cagcaacttc atgggctggt tcaggcaggc ccccggcaag 120 gagaggagg gcgtggccgc catgatgacc aagaacaaca acacctacta cgccgacagc 180 gtgaagggca ggttcaccat cagccacgac aacgccaaga acaccgtgta cctgcagatg 240 gacagcctga agcccgagga caccgccgtg tactactgcg ccgccgtgta caggaccagg 300 aggctgaggg tgctggaggc cgccaacttc gactactggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 179 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 179 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcaccg ccagcggcta caccagcagc aggtactgca tgggctggtt caggcagacc 120 cccggcaaga agagggaggg cgtggccgcc atctacaccg gcggcggcac caccttctac 180 cacggcagcg tgaagggcag gttcaccatc agccaggaca acaccaccaa caccgtgtac 240 ctgcagatgc acaacctgaa gcccgaggac accgccatgt actactgcgc cgccggcccc 300 gtgaccaggg cctgcgacga gtacaactac tggggccagg gcacccaggt gaccgtgagc 360 agc 363 <210> 180 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 180 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg gcagcggcta cagcctgagc aactactgca tgggctggtt caggcaggcc 120 cccggccagg gcagggaggg cgtggccagc ctgaggttcg tgagcggcgc caccttctac 180 gccgacagcg tgaagggcag gttcaccatc gcccaggaca acgccaagaa caccctgtac 240 ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgg catcaagagc 300 aggggcatct gcggcggcag gctggtggac gtggacttcg gcaactgggg ccagggcacc 360 caggtgaccg tgagcagc 378 <210> 181 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 181 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg ccagcggcta cagcatcaac aggatgggct ggttcaggca ggcccccggc 120 aaggagaggg agggcgtggc cgccatcagc atcggcggcg gccagaccta ctacgccgac 180 agcgtgaagg gcaggttcac catcagccag gacaacgcca agaacaccgt ggacctgcag 240 atgaacagcc tgaagcccga ggacaccgcc atgtactact gcgccgccgg cctggtgtac 300 ggcgaggcct ggctggacag caggcactac aacaagtggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 182 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 182 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcgcc aaggacggca tcaccatcca cgccgacagc 180 gtgaagggca ggttcaccat cagcaaggac aacgccaaga acaccctgta cctgcagatg 240 aacagcctga agcccgagga caccgccatg tactactgcg ccgcccacag gccctacggc 300 ccccccctga accccaggtg gtacacctac tggggccagg gcacccaggt gaccgtgagc 360 agc 363 <210> 183 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 183 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg ccagcggcta cacctacagc agctactgca tggcctggtt caggcaggcc 120 cccggcaagg agagggaggg cgtggccgcc atcgacagcg acggcagcac cagctacgcc 180 gacagcgtga agggcaggtt caccatcagc aaggacaacg ccaagaacac cctgtacctg 240 cagatgaaca gcctgaagcc cgaggacacc gccatgtact actgcgccgc cgccagcggc 300 aggtgcctgg gccccggcat caggagcctg atctggggcc agggcaccca ggtgaccgtg 360 agcagc 366 <210> 184 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 184 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcacc aaggacatca ccatccacgc cgacagcgtg 180 aagggcaggt tcaccatcag caaggacaac gccaagaaca ccctgtacct gcagatgaac 240 agcctgaagc ccgaggacac cgccatgtac tactgcgccg cccacaggcc ctacggcccc 300 cccctgaacc ccaggtggta cacctactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 185 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 185 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcccc accgacggca tcaccatcca cgccgacagc 180 gtgaagggca ggttcaccat cagcaaggac aacgccaaga acaccctgta cctgcagatg 240 aacagcctga agcccgagga caccgccatg tactactgcg ccgcccacag gccctacggc 300 ccccccctga accccaggtg gtacacctac tggggccagg gcacccaggt gaccgtgagc 360 agc 363 <210> 186 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 186 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcgcc aaggacggca tcaccatcca cgccgacagc 180 gtgaagggca ggttcaccat cagcaaggac aacgccaaga acaccctgta cctgcagatg 240 agcagcctga agcccgagga caccgccatg tactactgcg ccgcccacag gccctacggc 300 ccccccctga accccaggtg gtacacctac tggggccagg gcacccaggt gaccgtgagc 360 agc 363 <210> 187 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 187 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcggc aaggacggca tcaccatcca cgccgacagc 180 gtgaagggca ggttcaccat cagcaaggac aacgccaaga acaccctgta cctgcagatg 240 aacagcctga agcccgagga caccgccatg tactactgcg ccgcccacag gccctacggc 300 ccccccctga accccaggtg gtacacctac tggggccagg gcacccaggt gaccgtgagc 360 agc 363 <210> 188 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 188 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcacc aaggacatca ccatccacgc cgacagcgtg 180 aagggcaggt tcaccatcag caaggacaac gccaagaaca ccctgtacct gcagatgaac 240 agcctgaggc ccgaggacac cgccatgtac tactgcgccg cccacaggcc ctacggcccc 300 cccctgaacc ccaggtggta cacctactgg ggccagggca cccaggtgac cgtgagcagc 360 <210> 189 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 189 caggtgcagc tgcaggagag cggcggcggc agcgtgcaga ccggcggcag cctgaggctg 60 agctgcgccg ccagcggcta cagcatcaac aggatggcct ggttcaggca ggcccccggc 120 aaggagaggg agggcgtggc cgccatcagc atcggcggcg acaggaccta ctacgccgac 180 agcgtgaagg gcaggttcac catcagccag gacaacgcca agcacaccgt ggacctgcag 240 atgaacagcc tgaagcccga ggacaccgcc atgtactact gcgccgccgg cctggtgtac 300 ggcgaggcct ggctggacag caggcactac aacaagtggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 190 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 190 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg ccagcggcta cagcatcaac aggatgggct ggttcaggca ggcccccggc 120 aaggagaggg agggcgtggc cgccatcagc atcggcggcg gcaggaccta ctacgccgac 180 agcgtgaagg gcaggttcac catcagccag gacaacgcca agaacaccgt ggacctgcag 240 atgaacagcc tgaagcccga ggacaccgcc atgtactact gcgccgccgg cctggtgtac 300 ggcgaggcct ggctggacag caggcactac aacaagtggg gccagggcac ccaggtgacc 360 gtgagcagc 369 <210> 191 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 191 caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60 agctgcgccg tgagcggcga cagcacctac agcatgggct ggttcaggca gccccccggc 120 aaggagaggg agggcgtggc cgccatcacc aaggacggca tcaccatcca cgccgacagc 180 gtgaagggca ggttcaccat cagcggcgac aacgccaaga acaccctgta cctgcagatg 240 aacaacctga agcccgagga caccgccatg tactactgcg ccgcccacag gccctacggc 300 ccccccctga accccaggtg gtacacctac tggggccagg gcacccaggt gaccgtgagc 360 agc 363 <210> 192 <211> 484 <212> PRT <213> Homo sapiens <400> 192 Gln Gly Ser Ala Gly Pro Leu Gln Cys Tyr Gly Val Gly Pro Leu Gly 1 5 10 15 Asp Leu Asn Cys Ser Trp Glu Pro Leu Gly Asp Leu Gly Ala Pro Ser 20 25 30 Glu Leu His Leu Gln Ser Gln Lys Tyr Arg Ser Asn Lys Thr Gln Thr 35 40 45 Val Ala Val Ala Ala Gly Arg Ser Trp Val Ala Ile Pro Arg Glu Gln 50 55 60 Leu Thr Met Ser Asp Lys Leu Leu Val Trp Gly Thr Lys Ala Gly Gln 65 70 75 80 Pro Leu Trp Pro Pro Val Phe Val Asn Leu Glu Thr Gln Met Lys Pro 85 90 95 Asn Ala Pro Arg Leu Gly Pro Asp Val Asp Phe Ser Glu Asp Asp Pro 100 105 110 Leu Glu Ala Thr Val His Trp Ala Pro Pro Thr Trp Pro Ser His Lys 115 120 125 Val Leu Ile Cys Gln Phe His Tyr Arg Arg Cys Gln Glu Ala Ala Trp 130 135 140 Thr Leu Leu Glu Pro Glu Leu Lys Thr Ile Pro Leu Thr Pro Val Glu 145 150 155 160 Ile Gln Asp Leu Glu Leu Ala Thr Gly Tyr Lys Val Tyr Gly Arg Cys 165 170 175 Arg Met Glu Lys Glu Glu Asp Leu Trp Gly Glu Trp Ser Pro Ile Leu 180 185 190 Ser Phe Gln Thr Pro Pro Ser Ala Pro Lys Asp Val Trp Val Ser Gly 195 200 205 Asn Leu Cys Gly Thr Pro Gly Gly Glu Glu Pro Leu Leu Leu Trp Lys 210 215 220 Ala Pro Gly Pro Cys Val Gln Val Ser Tyr Lys Val Trp Phe Trp Val 225 230 235 240 Gly Gly Arg Glu Leu Ser Pro Glu Gly Ile Thr Cys Cys Cys Ser Leu 245 250 255 Ile Pro Ser Gly Ala Glu Trp Ala Arg Val Ser Ala Val Asn Ala Thr 260 265 270 Ser Trp Glu Pro Leu Thr Asn Leu Ser Leu Val Cys Leu Asp Ser Ala 275 280 285 Ser Ala Pro Arg Ser Val Ala Val Ser Ser Ile Ala Gly Ser Thr Glu 290 295 300 Leu Leu Val Thr Trp Gln Pro Gly Pro Gly Glu Pro Leu Glu His Val 305 310 315 320 Val Asp Trp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val 325 330 335 Arg Leu Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr 340 345 350 Val Gly Val Pro Tyr Arg Ile Thr Val Thr Ala Val Ser Ala Ser Gly 355 360 365 Leu Ala Ser Ala Ser Ser Val Trp Gly Phe Arg Glu Glu Leu Ala Pro 370 375 380 Leu Val Gly Pro Thr Leu Trp Arg Leu Gln Asp Ala Pro Pro Gly Thr 385 390 395 400 Pro Ala Ile Ala Trp Gly Glu Val Pro Arg His Gln Leu Arg Gly His 405 410 415 Leu Thr His Tyr Thr Leu Cys Ala Gln Ser Gly Thr Ser Pro Ser Val 420 425 430 Cys Met Asn Val Ser Gly Asn Thr Gln Ser Val Thr Leu Pro Asp Leu 435 440 445 Pro Trp Gly Pro Cys Glu Leu Trp Val Thr Ala Ser Thr Ile Ala Gly 450 455 460 Gln Gly Pro Pro Gly Pro Ile Leu Arg Leu His Leu Pro Asp Asn Thr 465 470 475 480 Leu Arg Trp Lys <210> 193 <211> 623 <212> PRT <213> Mus sp. <400> 193 Met Asn Arg Leu Arg Val Ala Arg Leu Thr Pro Leu Glu Leu Leu Leu 1 5 10 15 Ser Leu Met Ser Leu Leu Leu Gly Thr Arg Pro His Gly Ser Pro Gly 20 25 30 Pro Leu Gln Cys Tyr Ser Val Gly Pro Leu Gly Ile Leu Asn Cys Ser 35 40 45 Trp Glu Pro Leu Gly Asp Leu Glu Thr Pro Pro Val Leu Tyr His Gln 50 55 60 Ser Gln Lys Tyr His Pro Asn Arg Val Trp Glu Val Lys Val Pro Ser 65 70 75 80 Lys Gln Ser Trp Val Thr Ile Pro Arg Glu Gln Phe Thr Met Ala Asp 85 90 95 Lys Leu Leu Ile Trp Gly Thr Gln Lys Gly Arg Pro Leu Trp Ser Ser 100 105 110 Val Ser Val Asn Leu Glu Thr Gln Met Lys Pro Asp Thr Pro Gln Ile 115 120 125 Phe Ser Gln Val Asp Ile Ser Glu Glu Ala Thr Leu Glu Ala Thr Val 130 135 140 Gln Trp Ala Pro Pro Val Trp Pro Pro Gln Lys Val Leu Ile Cys Gln 145 150 155 160 Phe Arg Tyr Lys Glu Cys Gln Ala Glu Thr Trp Thr Arg Leu Glu Pro 165 170 175 Gln Leu Lys Thr Asp Gly Leu Thr Pro Val Glu Met Gln Asn Leu Glu 180 185 190 Pro Gly Thr Cys Tyr Gln Val Ser Gly Arg Cys Gln Val Glu Asn Gly 195 200 205 Tyr Pro Trp Gly Glu Trp Ser Ser Pro Leu Ser Phe Gln Thr Pro Phe 210 215 220 Leu Asp Pro Glu Asp Val Trp Val Ser Gly Thr Val Cys Glu Thr Ser 225 230 235 240 Gly Lys Arg Ala Ala Leu Leu Val Trp Lys Asp Pro Arg Pro Cys Val 245 250 255 Gln Val Thr Tyr Thr Val Trp Phe Gly Ala Gly Asp Ile Thr Thr Thr 260 265 270 Gln Glu Glu Val Pro Cys Cys Lys Ser Pro Val Pro Ala Trp Met Glu 275 280 285 Trp Ala Val Val Ser Pro Gly Asn Ser Thr Ser Trp Val Pro Pro Thr 290 295 300 Asn Leu Ser Leu Val Cys Leu Ala Pro Glu Ser Ala Pro Cys Asp Val 305 310 315 320 Gly Val Ser Ser Ala Asp Gly Ser Pro Gly Ile Lys Val Thr Trp Lys 325 330 335 Gln Gly Thr Arg Lys Pro Leu Glu Tyr Val Val Asp Trp Ala Gln Asp 340 345 350 Gly Asp Ser Leu Asp Lys Leu Asn Trp Thr Arg Leu Pro Pro Gly Asn 355 360 365 Leu Ser Thr Leu Leu Pro Gly Glu Phe Lys Gly Gly Val Pro Tyr Arg 370 375 380 Ile Thr Val Thr Ala Val Tyr Ser Gly Gly Leu Ala Ala Ala Pro Ser 385 390 395 400 Val Trp Gly Phe Arg Glu Glu Leu Val Pro Leu Ala Gly Pro Ala Val 405 410 415 Trp Arg Leu Pro Asp Asp Pro Pro Gly Thr Pro Val Val Ala Trp Gly 420 425 430 Glu Val Pro Arg His Gln Leu Arg Gly Gln Ala Thr His Tyr Thr Phe 435 440 445 Cys Ile Gln Ser Arg Gly Leu Ser Thr Val Cys Arg Asn Val Ser Ser 450 455 460 Gln Thr Gln Thr Ala Thr Leu Pro Asn Leu His Leu Gly Ser Phe Lys 465 470 475 480 Leu Trp Val Thr Val Ser Thr Val Ala Gly Gln Gly Pro Pro Gly Pro 485 490 495 Asn Leu Ser Leu His Leu Pro Asp Asn Arg Ile Arg Trp Lys Ala Leu 500 505 510 Pro Trp Phe Leu Ser Leu Trp Gly Leu Leu Leu Met Gly Cys Gly Leu 515 520 525 Ser Leu Ala Ser Thr Arg Cys Leu Gln Ala Arg Cys Leu His Trp Arg 530 535 540 His Lys Leu Leu Pro Gln Trp Ile Trp Glu Arg Val Pro Asp Pro Ala 545 550 555 560 Asn Ser Asn Ser Gly Gln Pro Tyr Ile Lys Glu Val Ser Leu Pro Gln 565 570 575 Pro Pro Lys Asp Gly Pro Ile Leu Glu Val Glu Glu Val Glu Leu Gln 580 585 590 Pro Val Val Glu Ser Pro Lys Ala Ser Ala Pro Ile Tyr Ser Gly Tyr 595 600 605 Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu Gly Leu Leu Val 610 615 620 <210> 194 <211> 486 <212> PRT <213> Mus sp. <400> 194 Thr Arg Pro His Gly Ser Pro Gly Pro Leu Gln Cys Tyr Ser Val Gly 1 5 10 15 Pro Leu Gly Ile Leu Asn Cys Ser Trp Glu Pro Leu Gly Asp Leu Glu 20 25 30 Thr Pro Pro Val Leu Tyr His Gln Ser Gln Lys Tyr His Pro Asn Arg 35 40 45 Val Trp Glu Val Lys Val Pro Ser Lys Gln Ser Trp Val Thr Ile Pro 50 55 60 Arg Glu Gln Phe Thr Met Ala Asp Lys Leu Leu Ile Trp Gly Thr Gln 65 70 75 80 Lys Gly Arg Pro Leu Trp Ser Ser Val Ser Val Asn Leu Glu Thr Gln 85 90 95 Met Lys Pro Asp Thr Pro Gln Ile Phe Ser Gln Val Asp Ile Ser Glu 100 105 110 Glu Ala Thr Leu Glu Ala Thr Val Gln Trp Ala Pro Pro Val Trp Pro 115 120 125 Pro Gln Lys Val Leu Ile Cys Gln Phe Arg Tyr Lys Glu Cys Gln Ala 130 135 140 Glu Thr Trp Thr Arg Leu Glu Pro Gln Leu Lys Thr Asp Gly Leu Thr 145 150 155 160 Pro Val Glu Met Gln Asn Leu Glu Pro Gly Thr Cys Tyr Gln Val Ser 165 170 175 Gly Arg Cys Gln Val Glu Asn Gly Tyr Pro Trp Gly Glu Trp Ser Ser 180 185 190 Pro Leu Ser Phe Gln Thr Pro Phe Leu Asp Pro Glu Asp Val Trp Val 195 200 205 Ser Gly Thr Val Cys Glu Thr Ser Gly Lys Arg Ala Ala Leu Leu Val 210 215 220 Trp Lys Asp Pro Arg Pro Cys Val Gln Val Thr Tyr Thr Val Trp Phe 225 230 235 240 Gly Ala Gly Asp Ile Thr Thr Thr Gln Glu Glu Val Pro Cys Cys Lys 245 250 255 Ser Pro Val Pro Ala Trp Met Glu Trp Ala Val Val Ser Pro Gly Asn 260 265 270 Ser Thr Ser Trp Val Pro Pro Thr Asn Leu Ser Leu Val Cys Leu Ala 275 280 285 Pro Glu Ser Ala Pro Cys Asp Val Gly Val Ser Ser Ala Asp Gly Ser 290 295 300 Pro Gly Ile Lys Val Thr Trp Lys Gln Gly Thr Arg Lys Pro Leu Glu 305 310 315 320 Tyr Val Val Asp Trp Ala Gln Asp Gly Asp Ser Leu Asp Lys Leu Asn 325 330 335 Trp Thr Arg Leu Pro Pro Gly Asn Leu Ser Thr Leu Leu Pro Gly Glu 340 345 350 Phe Lys Gly Gly Val Pro Tyr Arg Ile Thr Val Thr Ala Val Tyr Ser 355 360 365 Gly Gly Leu Ala Ala Ala Pro Ser Val Trp Gly Phe Arg Glu Glu Leu 370 375 380 Val Pro Leu Ala Gly Pro Ala Val Trp Arg Leu Pro Asp Asp Pro Pro 385 390 395 400 Gly Thr Pro Val Val Ala Trp Gly Glu Val Pro Arg His Gln Leu Arg 405 410 415 Gly Gln Ala Thr His Tyr Thr Phe Cys Ile Gln Ser Arg Gly Leu Ser 420 425 430 Thr Val Cys Arg Asn Val Ser Ser Gln Thr Gln Thr Ala Thr Leu Pro 435 440 445 Asn Leu His Leu Gly Ser Phe Lys Leu Trp Val Thr Val Ser Thr Val 450 455 460 Ala Gly Gln Gly Pro Pro Gly Pro Asn Leu Ser Leu His Leu Pro Asp 465 470 475 480 Asn Arg Ile Arg Trp Lys 485 <210> 195 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 195 His His His His His His 1 5 <210> 196 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 8xHis tag <400> 196 His His His His His His His His 1 5 <210> 197 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> SITE <222> (1)..(40) <223> This sequence may encompass 1-10 "Gly Gly Gly Ser" repeating units <400> 197 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Gly Ser 35 40 <210> 198 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> SITE <222> (1)..(50) <223> This sequence may encompass 1-10 "Gly Gly Gly Ser Gly" repeating units <400> 198 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 15 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 20 25 30 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 35 40 45 Ser Gly 50 <210> 199 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> SITE <222> (1)..(50) <223> This sequence may encompass 1-10 "Gly Gly Gly Gly Ser" repeating units <400> 199 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50 <210> 200 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> SITE <222> (1)..(30) <223> This sequence may encompass 1-10 "Gly Gly Ser" repeating units <400> 200 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 20 25 30 <210> 201 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> SITE <222> (1)..(40) <223> This sequence may encompass 1-10 "Gly Gly Ser Gly" repeating units <400> 201 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 20 25 30 Gly Gly Ser Gly Gly Gly Ser Gly 35 40 <210> 202 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> SITE <222> (1)..(6) <223> This sequence may encompass 3-6 residues <400> 202 His His His His His His 1 5

Claims

1. An isolated nucleic acid encoding an IL27Rα-binding molecule that specifically binds to the extracellular domain of interleukin-27 receptor alpha (IL27Rα), wherein the IL27Rα-binding molecule is as follows: Complementarity Determination Region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:50, CDR2 containing the amino acid sequence of SEQ ID NO:51, and CDR3 containing the amino acid sequence of SEQ ID NO:52; or CDR1 containing the amino acid sequence of SEQ ID NO:26, CDR2 containing the amino acid sequence of SEQ ID NO:27, and CDR3 containing the amino acid sequence of SEQ ID NO:28; or CDR1 containing the amino acid sequence of SEQ ID NO:68, CDR2 containing the amino acid sequence of SEQ ID NO:69, and CDR3 containing the amino acid sequence of SEQ ID NO:70; or CDR1 containing the amino acid sequence of SEQ ID NO:80, CDR2 containing the amino acid sequence of SEQ ID NO:81, and CDR3 containing the amino acid sequence of SEQ ID NO:82; or CDR1 contains the amino acid sequence of SEQ ID NO:86, CDR2 contains the amino acid sequence of SEQ ID NO:87, and CDR3 contains the amino acid sequence of SEQ ID NO:

88. Isolated nucleic acids containing a single-domain antibody.

2. The IL27Rα binding molecule is V H The isolated nucleic acid according to claim 1, wherein H.

3. The isolated nucleic acid according to claim 1, comprising a single-domain antibody that is humanized or a CDR grafted onto a heterologous framework.

4. The isolated nucleic acid according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 50, CDR2 comprises the amino acid sequence of SEQ ID NO: 51, and CDR3 comprises the amino acid sequence of SEQ ID NO:

52.

5. The isolated nucleic acid according to claim 4, comprising a single-domain antibody containing the amino acid sequence of SEQ ID NO:

10.

6. The isolated nucleic acid according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:26, CDR2 comprises the amino acid sequence of SEQ ID NO:27, and CDR3 comprises the amino acid sequence of SEQ ID NO:

28.

7. The isolated nucleic acid according to claim 6, wherein the single-domain antibody comprises the amino acid sequence of SEQ ID NO:

2.

8. The isolated nucleic acid according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:68, CDR2 comprises the amino acid sequence of SEQ ID NO:69, and CDR3 comprises the amino acid sequence of SEQ ID NO:

70.

9. The isolated nucleic acid according to claim 8, wherein the single-domain antibody comprises the amino acid sequence of SEQ ID NO:

16.

10. The isolated nucleic acid according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:80, CDR2 comprises the amino acid sequence of SEQ ID NO:81, and CDR3 comprises the amino acid sequence of SEQ ID NO:

82.

11. The isolated nucleic acid according to claim 10, comprising a single-domain antibody containing the amino acid sequence of SEQ ID NO:

20.

12. The isolated nucleic acid according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:86, CDR2 comprises the amino acid sequence of SEQ ID NO:87, and CDR3 comprises the amino acid sequence of SEQ ID NO:

88.

13. The isolated nucleic acid according to claim 12, comprising a single-domain antibody containing the amino acid sequence of SEQ ID NO:

22.

14. An expression vector comprising an isolated nucleic acid according to any one of claims 1 to 13.

15. Isolated cells comprising the expression vector according to claim 14.

16. A method for producing an IL27Rα-binding molecule that specifically binds to the extracellular domain of interleukin-27 receptor alpha (IL27Rα) from the cells described in Claim 15, comprising the step of culturing the cells to express the IL27Rα-binding molecule.

17. A pharmaceutical composition for treating a subject that requires such treatment, (i) an interleukin-27 receptor alpha (IL27Rα) binding molecule encoded by an isolated nucleic acid according to any one of claims 1 to 13, or (ii) The nucleic acid A pharmaceutical composition containing [the specified substance].

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