GP130-binding molecule and method of use

Abstract

This provides a gp130 binding molecule. [Solution] This disclosure provides a biologically active molecule comprising a single-domain antibody (sdAb) that specifically binds to the extracellular domain of human gp130, a composition comprising such an antibody, and a method of using the same.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 061,562, filed August 5, 2020; U.S. Provisional Application No. 63 / 078,745, filed September 15, 2020; and U.S. Provisional Application No. 63 / 135,884, filed 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 gp130, compositions comprising such single - domain antibodies, and methods of using the same.

Background Art

[0003] Background Glycoprotein 130 (gp130), also known as IL6ST, IL6R - β, and CDw130m, is a highly conserved transmembrane protein that forms a subunit of the type I cytokine receptor within the IL6 receptor family. Gp130 is ubiquitously expressed in the human body and is a common receptor subunit for at least nine cytokines including IL6, IL27, cardiotrophin - 1 (CT - 1) and neuropoietin (NP), oncostatin M (OSM), leukemia inhibitory factor (LIF), IL11, ciliary neurotrophic factor (CNTF) and cardiotrophin - like cytokine (CLC). These various cytokines mediate extremely diverse biological processes, and thus, gp130 has been shown to play roles in various types of cancer, cardiovascular diseases, and autoimmune diseases. By forming appropriate receptor complexes, gp130 can interact with various cytokines and as a result, is associated with more than 50 different cellular responses.

[0004] ​​The extracellular domain of gp130 contains six consecutive sandwich domains (D1--D6): five fibronectin type III domains (D2-D6) following the N-terminal immunoglobulin-like domain (D1). Most ligands interact with gp130 in the membrane-proximal domains D1, D2, and D3. The N-terminal D1 immunoglobulin-like domain is required for cytokine responses. In response to ligand binding, the intracellular domain interacts with Janus kinases (JAKs) to initiate intracellular signaling.

[0005] In addition to the membrane-bound form, there are three soluble isoforms of gp130, sgp130-RAPS, sgp130-E10, and "full-length" sgp130. Soluble forms of gp130 lack the transmembrane and cytoplasmic domains but retain the extracellular ligand-binding domain. Soluble forms of gp130 interact with the soluble form of the IL6 receptor. The physiological role of sgp130 has not been fully elucidated, but it has been hypothesized that the role of sgp130 is anti-inflammatory by inhibiting gp130 signaling. sgp130 has high affinity (1 mM) for the IL6:sIL6R complex, which drives the pro-inflammatory and pro-atherogenic IL6 trans-signaling pathway. Binding of sgp130 to IL6:sIL6R neutralizes the IL6:sIL6R complex and reduces its inflammatory activity. In mice, recombinant sgp130 (sgp130Fc) has been associated with a protective effect in a mouse experimental model of atherosclerosis. Soluble gp130 has also been reported to inhibit the functions of IL-6, OSM, LIF, and CNTF.

[0006] gp130 is ubiquitously expressed in the human body, but its expression varies significantly among various organs and cell types, being more highly expressed in adult organs than in fetal tissues. Because gp130 is involved in multiple cytokine signaling pathways, differences in the expression of other receptor subunits are thought to contribute to distinct and broadly variable functions related to the ligands of the receptors in which gp130 is involved. For example, IL-6R is present at relatively high levels in the liver, neutrophils, and leukocytes. LIF receptors are highly expressed in the nervous and immune systems. IL-11R is highly expressed in T lymphocytes, atria, and aorta.

[0007] Due to its central role in cellular signaling across a range of tissue and organ types, gp130 has been proposed as a target for intervention in a wide variety of human disease states. For example, anti-gp130 antibodies have been proposed for modulating acute inflammatory responses associated with trauma, infection, and injury. Harrison, et al. (1996) British Journal of Haematology 95(3):443-451. Harrison et al. demonstrated that monoclonal antibodies against the extracellular domain of gp130 are effective in downregulating the acute IL-6 response in acute responses. Okamato et al. observed that anti-gp130 monoclonal antibodies can inhibit IL-6-induced HIV-1 expression in U1 cells, suggesting that blocking gp130 signaling may have therapeutic potential for treating HIV-1 infection. Okamato, et al. (1997) Biochemistry and Molecular Biology International 43(4):733-740.

[0008] Agents that downregulate or inhibit gp130 activity have demonstrated usefulness in various cancer types. Xu et al. reported an oral small molecule gp130 inhibitor for the treatment of ovarian cancer. Xu, et al (2013) Mol Cancer Ther; 12(6); 937-49. Martin et al. demonstrated that gp130 expression is associated with high-grade bladder cancer and that inhibiting gp130 with siRNA elicits a tumor-specific response. This suggests that gp130 blockade has therapeutic potential to control tumor growth. Martin, et al (2019) Mol Cancer Ther; 18(2):413-420. Furthermore, Burger et al. demonstrated that anti-gp130 antibodies are more effective than anti-IL6 antibodies in treating myeloma and that treatment with gp130 antibodies completely prevented the development of plasmacytoma. Burger, et al (2017) Haematologica 102(2): 381-390.

[0009] Monoclonal antibodies are the most widely used reagents for protein detection and quantification; however, their large size (approximately 150 kDa) can limit their use in assays involving several competing reagents for recognition of closely spaced epitopes. A unique class of immunoglobulins containing a heavy chain domain and lacking a light chain domain (commonly called "heavy chain" antibodies (HCAb)) is found in camelids, including dromedaries, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicuñas, and guanacos, as well as cartilaginous fish such as sharks. The isolated variable domain region of HCAb is known as VHH (an abbreviation for "variable-heavy-heavy," reflecting its structure) or Nanobody® (Ablynx). Single-domain VHH antibodies have the advantage of being small in size (approximately 12-14 kD), with a molecular weight about 1 / 10th that of conventional mammalian IgG class antibodies. This facilitates the binding of these VHH molecules to the gp130 antigenic determinant, which may be unreachable by conventional monoclonal IgG forms (Ingram et al., 2018). Furthermore, VHH single-domain antibodies often exhibit high thermal stability, which facilitates drug delivery to regions 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 Initiative]

[0010] This disclosure provides a polypeptide that specifically binds to gp130.

[0011] This disclosure provides a polypeptide that specifically binds to the extracellular domain of gp130.

[0012] This disclosure provides a gp130-binding molecule that specifically binds to the extracellular domain of gp130 (e.g., human gp130).

[0013] In some embodiments, the gp130-binding molecule includes a single-domain antibody (sdAb) that specifically binds to the extracellular domain of human gp130.

[0014] In some embodiments, the gp130-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.

[0015] In some embodiments, the gp130 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 respect to 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.

[0016] In some embodiments, the gp130-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 substitutions) or 100% identity with respect to any one polypeptide sequence of SEQ ID NO: 2-7, as shown in Table 1 below.

[0017] (Table 1) TIFF2026063000000001.tif186155

[0018] In some embodiments, the aforementioned set of CDRs is incorporated into a humanized VHH framework to form a "humanized" sdAb gp130-binding molecule.

[0019] Furthermore, this disclosure provides a method for a chemical or recombinant process to prepare the gp130-binding molecule of this disclosure.

[0020] Furthermore, this disclosure provides nucleic acids encoding gp130-binding molecules. Table 2 below shows examples of DNA sequences encoding gp130-binding molecules as described herein.

[0021] (Table 2) DNA sequences encoding VHH in Table 1 TIFF2026063000000002.tif247158

[0022] In some embodiments, ILRb is mouse gp130.

[0023] In some embodiments, the gp130-binding molecule comprises a single-domain antibody (sdAb) that specifically binds to the extracellular domain of mouse or rat gp130 (mgp130) or specifically binds to the extracellular domains of both human and mouse gp130.

[0024] In some embodiments, the gp130-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.

[0025] In some embodiments, the gp130 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 respect to 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.

[0026] In some embodiments, the gp130-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 single polypeptide sequence from SEQ ID NO: 26 to 74, as shown in Table 3 below.

[0027] (Table 3) TIFF2026063000000003.tif144161TIFF2026063000000004.tif234161TIFF2026063000000005.t if234161TIFF2026063000000006.tif234161TIFF2026063000000007.tif225161TIFF2026063000 000008.tif234161TIFF2026063000000009.tif234161TIFF2026063000000010.tif234161TIFF20 26063000000011.tif234161TIFF2026063000000012.tif225161TIFF2026063000000013.tif54161

[0028] In some embodiments, the aforementioned set of CDRs is incorporated into a humanized VHH framework to form a "humanized" sdAb gp130-binding molecule.

[0029] Furthermore, this disclosure provides a method for a chemical or recombinant process to prepare the gp130-binding molecule of this disclosure.

[0030] Furthermore, this disclosure provides nucleic acids encoding gp130 binding molecules. Table 4 below shows examples of DNA sequences encoding hgp130 binding molecules, as described in Table 3 above.

[0031] (Table 4) DNA sequences encoding VHH in Table 3 TIFF2026063000000014.tif249170TIFF2026063000000015.tif201170TIFF2026063000000016.tif201170 TIFF2026063000000017.tif201170TIFF2026063000000018.tif201170TIFF2026063000000019.tif192170 TIFF2026063000000020.tif201170TIFF2026063000000021.tif201170TIFF2026063000000022.tif201170 TIFF2026063000000023.tif201170TIFF2026063000000024.tif196170TIFF2026063000000025.tif196170

[0032] Furthermore, the Disclosure provides recombinant viral vectors and nonviral vectors comprising a nucleic acid encoding the gp130-binding molecule of the Disclosure or the CDR of the gp130-binding molecule of the Disclosure.

[0033] This disclosure further provides recombinant viral vectors and nonviral vectors comprising a host cell containing a gp130-binding molecule of this disclosure or a nucleic acid encoding a CDR of a gp130-binding molecule of this disclosure.

[0034] This disclosure further provides host cells comprising recombinant viral vectors and non-viral vectors, each comprising a nucleic acid encoding the gp130-binding molecule of this disclosure or the CDR of the gp130-binding molecule of this disclosure.

[0035] This disclosure further provides pharmaceutical formulations, including recombinant viral vectors and nonviral vectors, that include nucleic acids encoding the gp130 binding molecule of this disclosure, as well as methods of use thereof in the treatment or prevention of diseases, disorders, or conditions in mammalian subjects.

[0036] This disclosure further provides a kit containing the gp130 binding molecule of this disclosure.

[0037] In another aspect, the Disclosure provides constructs for targeted delivery of therapeutic agents to gp130 receptor-expressing cells, wherein a gp130-binding molecule is optionally conjugated to one or more therapeutic agents via a chemical linker or polypeptide linker. Furthermore, the Disclosure provides the aforementioned method of use in the treatment of a disease related to gp130 expression in a subject, comprising the step of administering to a subject requiring treatment a therapeutically effective amount of the gp130-binding molecule conjugated to a therapeutic agent, either alone or in combination with one or more further therapeutic agents. In some embodiments, the disease to be treated is a disease, disorder, or condition related to signaling from a receptor containing gp130. In some embodiments, the gp130-binding molecule of the Disclosure is useful in the treatment of diseases related to dysregulated T cell or B cell activity. In some embodiments, the gp130-binding molecule of the Disclosure is useful in the treatment of inflammatory and autoimmune diseases. In some embodiments, the gp130-binding molecules of this disclosure are useful in treating neoplastic diseases associated with abnormal cellular activity resulting from dysregulated signaling in gp130-expressing cells.

[0038] In another aspect, the Disclosure provides constructs for identifying gp130 receptor-expressing cells, wherein a gp130-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 gp130 receptor-expressing cells in a subject, comprising the steps of administering an effective amount of gp130 receptor conjugated to an imaging agent to a subject requiring treatment, and evaluating the subject for the presence of imaging agents conjugated to gp130-binding molecules.

[0039] In some embodiments, the gp130-binding molecule of this disclosure is useful for inhibiting interferon-γ activity in vitro and / or in vivo. In some embodiments, the IFNgR1-binding molecule of this disclosure is useful in the treatment of autoimmune diseases. Furthermore, this disclosure provides a method of the aforementioned use in the treatment of autoimmune diseases in a subject, comprising the step of administering a therapeutically effective amount of the gp130-binding molecule of this disclosure to the subject. In some embodiments, the gp130-binding molecule of this disclosure may be used alone or in combination with one or more adjunct therapeutic agents. In some embodiments, the disease to be treated is a disease, disorder, or condition related to signaling from receptors including gp130. In some embodiments, the gp130-binding molecule of this disclosure is useful in the treatment of diseases related to dysregulated T cell or B cell activity. In some embodiments, the gp130-binding molecule of this disclosure is useful in the treatment of autoimmune diseases. In some embodiments, the gp130-binding molecule of this disclosure is useful in the treatment of neoplasms. In some embodiments, the gp130-binding molecule of the Disclosure is administered to a subject in a pharmaceutically acceptable formulation. In some embodiments, the gp130-binding molecule of the Disclosure is administered to a subject by administering a composition comprising a recombinant viral vector or a non-viral vector containing a nucleic acid sequence encoding the gp130-binding molecule of the Disclosure.

[0040] In another aspect, the present disclosure provides a gp130-binding molecule modified to extend the duration of action in vivo, wherein the gp130-binding molecule is conjugated to one or more carrier molecules.

[0041] This disclosure provides a gp130-binding molecule comprising a polypeptide sequence that specifically binds to the extracellular domain of gp130, and a method for using it in the isolation, depletion, or enrichment of gp130-expressing cells in a biological sample. [Invention 1001] A gp130-binding molecule that specifically binds to the extracellular domain of gp130. [Invention 1002] A gp130-binding molecule according to the present invention 1001, containing a single-domain antibody (sdAb). [Invention 1003] sdAb is shown in the following table: A gp130-binding molecule of the present invention 1002, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3, as shown in the row of TIFF2026063000000026.tif63155. [Invention 1004] A gp130-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, 3, 4, 5, 6, and 7. [Invention 1005] sdAb is shown in the following table: A gp130-binding molecule of the present invention 1002, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3, as shown in the rows of TIFF2026063000000027.tif53165TIFF2026063000000028.tif245165TIFF2026063000000029.tif160165. [Invention 1006] The gp130-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: 26-74. [Invention 1007] A gp130-binding molecule according to either Invention 1003 or 1005, comprising a humanized or otherwise CDR grafted onto a heterogeneous framework. [Invention 1008] A gp130-binding molecule according to any one of the present invention 1001 to 1007, further comprising a labeling agent, an imaging agent, and / or a therapeutic agent. [Invention 1009] A method for treating or preventing a disease, disorder, or condition in a mammalian subject by administering a therapeutically effective amount of any gp130-binding molecule according to any of invention 1001 to 1008 or a pharmaceutically acceptable formulation thereof to a mammalian subject. [Invention 1010] The method of the present invention 1009, wherein the disease is a neoplastic disease. [Invention 1011] A gp130-binding molecule according to any of invention 1001-1008 for use in the isolation, depletion, or concentration of gp130+ cells from a biological sample. [Invention 1012] A nucleic acid sequence encoding any of the gp130 binding molecules according to invention 1001 to 1008. [Invention 1013] A recombinant viral vector or non-viral vector comprising the nucleic acid of the present invention 1012. [Invention 1014] A host cell containing the nucleic acid of the present invention 1012. [Invention 1015] A pharmaceutical formulation comprising a viral vector or a non-viral vector according to the present invention 1013. [Invention 1016] A kit comprising any of the gp130-binding molecules of Invention 1001 to 1008. [Modes for carrying out the invention]

[0042] Detailed description of the invention Introduction To facilitate a more readily understood 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] (Table 5) Abbreviations of amino acids TIFF2026063000000030.tif110128

[0050] 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).

[0051] definition Unless otherwise specified, the following terms are intended to have the meanings set forth below. Other terms are defined elsewhere throughout this specification.

[0052] 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.

[0053] 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.

[0054] 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., a gp130-binding molecule or engineered cells expressing a gp130-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.

[0055] 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].

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] gp130 cells: The terms “gp130 cells,” “gp130-expressing cells,” “gp130-positive cells,” and “gp130+” cells are used herein synonymously to refer to cells that express and show the gp130 antigen on the extracellular surface of the cell membrane. Similarly, the terms “gp130-negative cells” and “gp130- cells” are used herein synonymously to describe cells that do not express and show the gp130 antigen on their cell surface.

[0061] 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 an antibody or a grafted antibody or its variant 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 follows the Kabat numbering rules or a hybrid of the Kabat and Chothia numbering rules.

[0062] 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%.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Concentrated: As used herein, the term "concentrated" refers to a sample that has been non-naturally engineered such that (a) the species of interest (e.g., a molecule or a cell) is present at a concentration higher (e.g., at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold) than the concentration of that species in a starting sample, e.g., a biological sample (e.g., a sample in which the molecule is naturally present or a sample present after administration), or (b) the molecule is present at a concentration higher than the environment in which it was made (e.g., a recombinantly modified bacterial or mammalian cell).

[0067] Extracellular domain: As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is outside the plasma membrane of a cell. The cell surface protein may be a transmembrane protein, a cell surface protein, or a membrane-bound protein.

[0068] 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).

[0069] 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.

[0070] Combined with: As used herein, the term “in combination with” refers to the administration of a first agent and at least one further agent (i.e., a second, third, fourth, fifth, etc.) to a subject when used in relation to the administration of multiple agents to a subject. For the purposes of the present invention, an agent (e.g., a gp130 conjugate molecule) is considered to be administered in combination with a second agent (e.g., an immune checkpoint pathway modulator) if, at the time of administration of the first agent, the biological effect resulting from the administration of the first agent persists in the subject in such a way that the therapeutic effects of the first and second agents overlap. For example, PD1 immune checkpoint inhibitors (e.g., nivolumab or pembrolizumab) are typically administered by IV infusion every two or three weeks, whereas the gp130 conjugate molecules of this disclosure are typically administered more frequently, for example, daily, twice daily, or weekly. However, even if the first agent is administered at a considerable time (e.g., several days or weeks) after the administration of the second agent, the first agent (e.g., pembrolizumab) may provide a long-lasting therapeutic effect, and the second agent (e.g., a gp130 binding molecule) may provide a therapeutic effect for as long as the therapeutic effect of the first agent continues, so that the second agent is considered to have been administered in combination with the first agent. In one embodiment, an agent is considered to have been administered in combination with the second agent if the first and second agents are administered simultaneously (within 30 minutes of each other), concurrently, or consecutively. In some embodiments, the first agent is considered to have been administered "concurrently" with the second agent if the first and second agents are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other. The term “in combination with” is also understood to apply to situations in which a first agent and a second agent are co-prescribed in a single pharmaceutically acceptable formulation, and the co-formulation is administered to the subject. In certain embodiments, for example, when one agent is administered before one or more other agents, the gp130 conjugate molecule and the adjuvant are administered or applied sequentially. In other embodiments, the gp130 conjugate molecule and the adjuvant are administered simultaneously.For example, when two or more drugs are administered simultaneously or nearly simultaneously, the two or more drugs may be present in two or more separate formulations, or they may be combined to form a single formulation (i.e., a co-formulation). Regardless of whether the drugs are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0071] Treatment is required: As used herein, the term “requiring treatment” refers to a judgment made by a physician or other caregiver regarding a subject, indicating that the subject needs treatment or would potentially benefit from it. This judgment is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0072] Requires prevention: As used herein, the term “requiring prevention” refers to a judgment made by a physician or other caregiver regarding a subject, indicating that the subject needs or would potentially benefit from preventive care. This judgment is based on a variety of factors within the scope of the physician's or caregiver's expertise.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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).

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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).

[0082] 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%.

[0083] 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).

[0084] Prevent: As used herein, the terms “prevent,” “prevention,” and “prevention” refer to a course of action initiated with respect to a subject before the onset of a disease, disorder, condition, or its symptoms, in order to prevent, suppress, inhibit, or reduce the risk of the subject developing a disease, disorder, condition, or other condition, either temporarily or permanently (as confirmed by the absence of clinical symptoms, for example), or to delay the onset of such disease, disorder, condition, or other condition. A course of action to prevent a disease, disorder, or condition in a subject is typically applied in the context of a subject who is predisposed to developing a disease, disorder, or condition due to genetic, empirical, or environmental factors that contribute to the development of that particular disease, disorder, or condition. In certain specific cases, the terms “prevent,” “prevention,” and “prevention” are also used to mean delaying the progression of a disease, disorder, or condition from an existing state to a more harmful state.

[0085] 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.

[0086] 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.

[0087] 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 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 under 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 confirmed by flow cytometry, as described when it is confirmed by STAT (e.g., STAT1, STAT3, STAT5) phosphorylation levels according to methods well known in the art. For example, STAT5 phosphorylation may be measured using flow cytometry as described in Horta, et al. and Garcia, et al., and may also be measured using a commercially available kit, such as the Phospho-STAT5 (Tyr694) kit (commercially available from Perkin-Elmer, Waltham MA as part number 64AT5PEG), carried out in general accordance with the instructions provided by the manufacturer.

[0088] 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."

[0089] 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.

[0090] 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, if the ligand is gp130-binding sdAb and the receptor contains gp130, the equilibrium dissociation constant of gp130-binding sdAb / gp130 ECD is approximately 10 5Over M, or about 10 6 Over M, or about 10 7 Over M, or about 10 8 Over M, or about 10 9 Over M, or about 10 10 M or more, or about 10 11If the M value is greater than M, gp130-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 hgp130 isoforms (e.g., gp130-binding sdAb). The binding affinity of gp130-binding molecules to gp130 as used herein may be determined and / or quantified by surface plasmon resonance ("SPR"). When evaluating the binding affinity of gp130-binding molecules to gp130, 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: 281) or 8xHis (SEQ ID NO: 282)) for retention on the chip. In some embodiments, the gp130 binding molecule may be immobilized on the chip, and gp130 (or its ECD fragment) may be provided in the mobile phase. Alternatively, gp130 (or its ECD fragment) may be immobilized on the chip, and the gp130 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 a gp130-binding molecule to gp130 using SPR, the gp130-binding molecule may be derivatized by C-terminal addition of a polyHis sequence (e.g., 6xHis (SEQ ID NO:281) or 8xHis (SEQ ID NO:282)) and immobilized on an NTA derivatized sensor chip, and the hgp130 receptor subunit whose gp130 VHH binding affinity is being evaluated is provided in the mobile phase. Means for incorporating a polyHis sequence into the C-terminus of a gp130-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 a gp130-binding molecule to gp130 is evaluated using SPR in general accordance with the disclosure of examples.

[0091] 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.

[0092] 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.

[0093] To be suffering from: As used herein, the term “suffering from” refers to a physician’s decision regarding a subject based on available, objective or subjective information accepted in the field for identifying a disease, disorder, or condition, including but not limited to X-rays, CT scans, conventional diagnostic clinical tests (e.g., blood cell counts), genomic data, protein expression data, and immunohistochemistry, from which the subject needs or would benefit from treatment. The term “suffering from” is typically used in conjunction with a specific disease condition; for example, “suffering from a neoplasm” refers to a subject diagnosed with the presence of a neoplasm.

[0094] 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 gp130 isoforms, referred to synonymously with gp130 cells, gp130+ cells, gp130 T cells, or gp130+ T cells.

[0095] 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.

[0096] Therapeutic effective dose: As used herein, the phrase “therapeutic dose” refers to the amount of an agent that, when administered to a subject in a single dose alone, as part of a pharmaceutical composition or treatment regimen, or as part of a series of doses, produces a positive effect on any quantitative or qualitative symptom, aspect, or feature of a disease, disorder, or condition. The therapeutic dose can be determined by measuring the relevant physiological effects and may be adjusted in relation to the administration plan and in response to a diagnostic analysis of the subject’s condition. Parameters for evaluation to determine the therapeutic dose of an agent are determined by a physician using diagnostic criteria accepted in the art, including but not limited to age, weight, sex, overall health, ECOG score, observable physiological parameters, blood concentration, blood pressure, electrocardiogram, computed tomography, and X-ray features. Alternatively, or in addition to the above, other parameters commonly assessed in clinical practice may be monitored to determine whether a therapeutically effective dose of the drug has been administered to the subject, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptoms, aspects, or characteristics of the disease, disorder, or condition, biomarkers (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), decrease in serum tumor markers, improvement in the criteria for response to solid tumors (RECIST), improvement in immune-related response criteria (irRC), increase in overall survival, extension of progression-free survival, extension of progression-free survival, increase in treatment success time, extension of recurrence-free survival, extension of time to next treatment, improvement in objective response rate, improvement in duration of response, decrease in tumor burden, complete remission, partial remission, or stable condition. These parameters are determined by the clinician in the field to assess the improvement in the subject's condition in response to the administration of the drug. In one embodiment, a therapeutically effective dose refers to the amount of an agent that, when used alone or in combination with another agent, produces a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder, or condition, without causing any irreversible serious adverse events during the course of administration of the agent to a mammalian subject.

[0097] 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.

[0098] 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 gp130-conjugated 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).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] explanation This disclosure provides a gp130-binding molecule comprising a single-domain antibody that specifically binds to the extracellular domain of gp130. In some embodiments, gp130 is human gp130. In some embodiments, gp130 is mouse (or rat) gp130.

[0103] Human gp130 In one embodiment, it specifically binds to the extracellular domain of the human gp130 receptor subunit (hgp130). hgp130 contains a 22-amino acid N-terminal signal sequence, and is expressed as a 918-amino acid precursor, which is post-translationally cleaved to form an 896-amino acid mature protein. The classic full-long acid hgp130 precursor (including the signal peptide) has the following amino acid sequence: It is a 918-amino acid polypeptide containing TIFF2026063000000031.tif80134.

[0104] For the purposes of this disclosure, the amino acid residue numbering of the human gp130 polypeptide as described herein follows the numbering of this classical sequence (UniProt reference number P40189, SEQ ID NO:1). Amino acids 1-22 of SEQ ID NO:1 have been identified as the signal peptide of hgp130, amino acids 23-619 of SEQ ID NO:1 have been identified as the extracellular domain, amino acids 620-641 of SEQ ID NO:1 have been identified as the transmembrane domain, and amino acids 642-918 of SEQ ID NO:1 have been identified as the intracellular domain.

[0105] To produce antibodies that bind to the ECD of gp130, immunization can be performed using the extracellular domain of hgp130. The extracellular domain of hgp130 has the following sequence: This is a 597-amino acid polypeptide, TIFF2026063000000032.tif53134.

[0106] Mouse GP130 In one embodiment, it specifically binds to the extracellular domain of the mouse or rat gp130 receptor subunit (mgp130). mgp130 contains a 22-amino acid N-terminal signal sequence and is expressed as a 917-amino acid precursor that is post-translationally cleaved to form an 895-amino acid mature protein. The classic full-long acid mgp130 precursor (containing the 22-signal peptide) has the following amino acid sequence: It is a 917-amino acid polypeptide with the IP address TIFF2026063000000033.tif85134.

[0107] For the purposes of this disclosure, the numbering of amino acid residues of the mgp130 polypeptide as described herein follows the numbering of this classical sequence (UniProt reference number Q00560, SEQ ID NO: 279). Amino acids 1-22 of SEQ ID NO: 279 have been identified as the signal peptide of mgp130, amino acids 23-617 of SEQ ID NO: 279 as the extracellular domain, amino acids 618-639 of SEQ ID NO: 279 as the transmembrane domain, and amino acids 640-917 of SEQ ID NO: 279 as the intracellular domain.

[0108] To produce antibodies that bind to the ECD of gp130, immunization can be performed using the extracellular domain of mgp130. The extracellular domain of the mgp130 receptor has the following sequence: This is a 595-amino acid polypeptide, TIFF2026063000000034.tif53133.

[0109] Identification of gp130-binding molecules and single-domain antibodies In some embodiments, the gp130-binding molecule of this disclosure is a single-domain antibody (sdAb). This disclosure relates to gp130-binding molecules, including single-domain antibodies (sdAbs), that specifically bind to the extracellular domain of human gp130 isoforms (hgp130) found on all gp130-expressing cells.

[0110] 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. HGP130-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 hGP130 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) AntibodiesSeen in (Basel) 8(1). Alternatively, hgp130 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 hgp130 or an immunologically active fragment thereof. Hgp130-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 hgp130 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.

[0111] 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.

[0112] experiment The single-domain antibody of this disclosure was obtained from camels by immunization with the extracellular domain of the gp130 receptor. The gp130VHH molecule of this disclosure was prepared 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 gp130, the human IgG1 hinge domain domain, and the human IgG1 heavy chain Fc, produced by recombinant production using human gp130 and mouse gp130 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 sequences 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 gp130 (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. Gp130-binding molecules showing specific binding to the gp130 antigen were isolated, sequenced, and sequence-analyzed to identify VHH sequences, CDRs, and unique VHH chronotypes. As used herein, the term "chronotype" refers to a collection of binding molecules originating from the same B cell progenitor cell, 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 hgp130 ECD antigen (anti-human gp130VHH) and CDRs isolated from such VHH. Table 3 shows VHH molecules that specifically bound to the ngp130 ECD antigen (anti-mouse gp130VHH) 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.

[0113] To further fully characterize the binding properties of the VHH molecules prepared as described above and to evaluate their binding affinity, representative examples of each human and mouse 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 experiments are summarized in Tables 6 and 7 below.

[0114] (Table 6) Binding of anti-hGP130 monoFc VHH (ligand) to hGP130-his (Antigen: Sino Biological, Catalog No. 10974) TIFF2026063000000035.tif61155

[0115] (Table 7) Binding of anti-mGP130 monoFc VHH (ligand) to hGP130-his (Antigen: Sino Biological, Catalog No. 10974) TIFF2026063000000036.tif69155

[0116] As demonstrated by the data shown in Tables 6 and 7 above, the gp130-binding molecules prepared in accordance with the disclosure showed specific binding to the extracellular domain of gp130 and exhibited a certain range of affinity to the extracellular domain of gp130.

[0117] In some cases, due to sequence or structural similarities between the extracellular domains of gp130 receptors from various mammalian species, immunization with an antigen derived from gp130 of a first mammalian species (e.g., hgp130-ECD) may produce antibodies that specifically bind to gp130 receptors of one or more further mammalian species. Such antibodies are called "cross-reactive." For example, immunization of camelid animals with a human-derived antigen (e.g., hgp130-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 gp130 VHH" or "hgp130 VHH" merely indicates that the species of gp130 antigen used to immunize the camelid animals from which VHH originated was human gp130 (e.g., hgp130, ECD, SEQ ID NO: 278), and should not be understood as a limitation regarding the specific binding affinity of VHH to gp130 molecules of other mammalian species. Similarly, the use of the terms "mouse gp130 VHH" or "mgp130 VHH" merely indicates that the species of gp130 antigen used to immunize the camelid animals from which VHH originated was mouse gp130 (e.g., mgp130 ECD, SEQ ID NO: 280), and should not be understood as a limitation regarding the specific binding affinity of VHH to gp130 molecules of other mammalian species.

[0118] This disclosure provides a gp130-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:2-7.

[0119] This disclosure provides a gp130-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:26-74.

[0120] This disclosure provides gp130-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.

[0121] This disclosure provides gp130-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.

[0122] Modified single-domain antibodies CDR graft sdAb In some embodiments, the gp130-conjugated sdAb of this disclosure is a CDR-grafted gp130-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 a gp130-conjugated molecule comprising a CDR-grafted gp130-conjugated sdAb, the CDR-grafted gp130-conjugated sdAb comprising a set of CDRs 1, 2, and 3 as shown in the row in Table 1A above. In some embodiments, the Disclosure provides a gp130-binding molecule comprising a CDR-grafted gp130-binding sdAb, the CDR-grafted gp130-binding sdAb comprising a set of CDR1, 2, and 3 as shown in the row in Table 2 above.

[0123] Chimeras and humanized sdAbs Any framework region can be used with the CDR as described herein. In some embodiments, the gp130-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 gp130-conjugated sdAb derived from hgp130-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.

[0124] In some embodiments, V as described herein HH can be humanized to include the human framework domain. Humanized V H Examples of human germlines that can be used to produce H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).

[0125] Elimination of N-linked glycosylation sites In some embodiments, the amino acid sequence of gp130-linked sdAb (particularly the CDR sequence) 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 other than 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 gp130-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.

[0126] gp130 binding molecule containing further agents In some embodiments, the gp130-conjugated molecules of the present disclosure include a gp130 single-domain antibody (sdAb) conjugated to one or more further biologically active agents, including but not limited to combinations of therapeutic agents, chemically active agents, optically active agents, or radioactive materials. 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 gp130-conjugated sdAb, and this combination results in a gp130-conjugated molecule with further utility or different utility.

[0127] 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 gp130-binding molecules to specific cell or tissue types; therapeutic agents (e.g., therapeutic agents including small molecule or polypeptide agents); and agents that make the molecule more visible to optical or electromagnetic sensors (e.g., radionucleotides or fluorescent substances). In some embodiments, the linker may be a cleavable or non-cleavable linker. As intended herein, using a cleavable linker in the gp130-binding molecule facilitates the release of the therapeutic agent into the intracellular cytoplasm during the internal translocation of the gp130-binding molecule. Using an uncleavable linker would allow for the release of the gp130-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).

[0128] In some embodiments, the gp130-binding molecule comprises a gp130-binding sdAb stably bound to a further agent, linked via a linker. The linker is a covalent bond between the two elements of the gp130-binding molecule (e.g., hgp130-binding VHH and PEG polymer). The linker may be a covalent bond, a chemical linker, or a peptide linker. A suitable linker generally includes a “mobile linker” of sufficient length to move to some extent between the gp130-binding sdAb and the linked agent. Examples of chemical linkers include arylacetylenes, ethylene glycol oligomers containing 2-10 monomer units, diamines, dibasic acids, amino acids, or combinations thereof. In some embodiments, the linker is a peptide linker. A suitable peptide linker can be easily selected and may be a linker of any suitable length, for example, a peptide linker with one amino acid (e.g., Gly), two, three, four, five, six, seven, eight, nine, ten, ten-two, twenty-three, thirty-five, or more than fifty amino acids. Suitable peptide linkers are known in the art and include, for example, peptide linkers containing mobile amino acid residues such as glycine and serine. An example of a mobile linker is a glycine polymer (G). n These include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other mobile linkers. Glycine and glycine-serine polymers are relatively structurally indeterminate and therefore can serve as neutral tethers between components. Further examples of mobile linkers include glycine polymer (G) nThese include glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers. Glycine and glycine-serine polymers are relatively structurally indeterminate and therefore may serve as neutral tethers between components. 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 gp130-linked sdAb disclosed herein. In some embodiments, the linker has the formula (GGGS)n (SEQ ID NO:283), (GGGSG)n (SEQ ID NO:284), (GGGGS)n (SEQ ID NO:285), (GGS)nG (SEQ ID NO:286), or (GGSG)n (SEQ ID NO:287), where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0129] Immunomodulators In some embodiments, the gp130-conjugating molecules of this disclosure include immunomodulators (immunoconjugates). Immunomodulators that can be conjugated to the hgp130-conjugating sdAb of this 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 gp130 or cells expressing gp130.

[0130] Flag tag In one embodiment, the disclosure provides a gp130-conjugated molecule containing 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 gp130-conjugated sdAb polypeptide further comprises a C-terminal c-myc epitope tag.

[0131] Chelated peptides In one embodiment, the present disclosure provides a gp130-conjugated molecule comprising one or more transition metal chelated polypeptide sequences. As described in U.S. Patent No. 4,569,794 by Smith et al., issued February 11, 1986, the incorporation of such transition metal chelated domains facilitates purification by immobilized metal affinity chromatography (IMAC). Examples of transition metal chelated polypeptides useful in the implementation of the gp130-conjugated 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 chelated polypeptides useful in the implementation of the gp130-conjugated molecule are polypeptides comprising 3 to 6 consecutive histidine residues (SEQ ID NO: 288), such as 6-histidine (His)6 peptide (SEQ ID NO: 281), 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 hgp130-binding molecule to such a chelated peptide facilitates the targeted delivery of transition metal ions to gp130-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.

[0132] Carrier molecule In some embodiments, the gp130-conjugated 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 gp130-conjugated 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.

[0133] Water-soluble polymers In some embodiments, the gp130-linked sdAb is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the implementation of this gp130-linked molecule include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymer of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefin alcohol, polysaccharides, poly-α-hydroxy acids, polyvinyl alcohol (PVA), polyphosphoren, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0134] 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 attached to the polypeptide, respectively. In some embodiments, PEG may be attached to the sdAb directly or covalently (e.g., via a lysine side chain, a sulfhydryl group of cysteine, or an N-terminal amine), and optionally, a linker is used between the PEG and the sdAb. In some embodiments, the gp130-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.

[0135] In some cases, the gp130-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. As a result, when PEGylating the gp130-binding molecules of this disclosure, particularly when aldehyde chemistry is used, position 1 of the gp130-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 gp130 binding molecule of this disclosure includes an amino acid substitution selected from the group of Q1E and Q1D.

[0136] 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.

[0137] The molecular weight of PEG used in the gp130-binding molecule is not limited to any particular range. The molecular weight of the PEG component of the gp130-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 gp130 binding molecule, the PEG is a 40kD branched PEG containing two 20kD arms.

[0138] This disclosure also intends gp130-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 gp130-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.

[0139] 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.

[0140] 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.

[0141] PEG can be bound to the gp130 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 the succinate ester of polyethylene glycol with N-hydroxysuccinilimide.

[0142] 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.

[0143] The PEG portion of the PEGylated gp130-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.

[0144] 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 format is used to facilitate the expression of a heterodimer polypeptide conjugate, specifically a first polypeptide (e.g., gp130-bound sdAb) on a first Fc monomer having a "knob" modification, and a second polypeptide on a second Fc monomer having a "hole" modification.

[0145] Targeted domains In some embodiments, the gp130-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 gp130-binding sdAb sequence of the fusion protein and the targeting domain sequence, in order to facilitate selective binding to a specific cell type or tissue expressing a cell surface molecule that specifically binds to the polypeptide sequence ("targeting domain").

[0146] In some embodiments, a gp130-binding molecule can be targeted to a specific cell type by functionally linking it to a targeting domain for the gp130-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 a gp130+ T cell.

[0147] 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.

[0148] In another aspect, the present disclosure provides a polyvalent binding molecule comprising (a) a gp130 binding molecule and (b) a second binding molecule that specifically binds to the extracellular domain of a second cell surface molecule, wherein the gp130 binding molecule and the second binding molecule are functionally linked, optionally via a chemical linker or polypeptide linker. In some embodiments, the gp130 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. According to the disclosure by Gonzalez et al., the second binding molecule specifically binds to (i) a component of a cytokine receptor that activates the cellular JAK / STAT pathway, other than a receptor in which gp130 forms a signaling complex in response to its native ligand; (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).

[0149] 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).

[0150] In some embodiments, the targeting domain of the gp130-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 gp130-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.

[0151] 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.

[0152] sign In some embodiments, the gp130-binding molecule of this disclosure is functionally linked to one or more labels. In some embodiments, the labels are incorporated to facilitate use as imaging agents, diagnostic agents, 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, a gp130-binding sdAb (e.g., gp130-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 gp130-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.

[0153] Therapeutic drugs In some embodiments, the gp130-binding molecules of this disclosure are functionally linked to therapeutic agents. Examples of therapeutic agents include therapeutic small molecules (e.g., chemotherapeutic agents) or biotherapeutic agents, such as antibodies, cytotoxic or cell division-arresting compounds, radioisotopes, plant-derived, fungal or bacterial-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 coating proteins), therapeutic antibodies, and chemotherapeutic agents, as will be further fully described herein.

[0154] In some embodiments, the therapeutic agent functionally linked to the gp130 binding molecule of this disclosure is, for example, a short-range radiation emitter including a short-range, high-energy α- emitter. Examples of such radioisotopes include α- emitters, β- emitters, γ- emitters, or β / γ- emitters. Radioisotopes useful as therapeutic agents include yttrium-90( 90 Y), Lutetium-177( 177 Lu), Actinium-225 ( 225 Ac), Astatine-211 ( 211 at), Rhenium-186( 186 Re), Bismuth-212 212 Bi), Bismuth-213 213Bi), and Rhodium-188( 188 It contains Rh.

[0155] In some embodiments, the gp130-binding molecule is functionally linked to a cytotoxic agent (or its derivatives), such as mytansinol or DM1 mytansinoid, taxane, or calitiamycin, Pseudomonas exotoxin A, debuganin, lysine toxin, diphtheria toxin, amatoxin, e.g., α-amanitin, saporin, mytansin, mytansinoid, auristatin, anthracycline, calitiamycin, irinotecan, SN-38, duocalmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer, or a variant thereof.

[0156] Synthesis of gp130 binding molecules: In some embodiments, the gp130-binding molecule of this disclosure is a polypeptide. However, in some embodiments, only a portion of the gp130-binding molecule is a polypeptide, for example, the gp130-binding molecule includes a non-peptidyl domain (e.g., PEG-gp130-binding sdAb conjugate, radionucleotide-gp130-binding sdAb conjugate, or small molecule-gp130-binding sdAb conjugate). The following provides guidance to enable solid-phase and recombinant synthesis of the polypeptide portion (domain) of the gp130-binding molecule of this disclosure. In embodiments where only a portion of the gp130-binding molecule is a polypeptide, the peptidyl domain of the gp130-binding molecule will be understood as a process intermediate that may undergo further processing to complete the synthesis of the desired gp130-binding molecule. The polypeptide domain of the gp130-binding molecule may be produced by conventional methodologies for polypeptide construction, including recombinant or solid-phase synthesis, as described in more detail below.

[0157] 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 gp130-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 gp130-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).

[0158] In some embodiments, the polypeptide domain of the gp130-binding molecule of this disclosure can be produced by chemical synthesis. The chemical synthesis of the polypeptide domain of the gp130-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 gp130-binding molecule of this disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, α-functional groups and any reactive side chains may be protected by acid-unstable or basic-acid-unstable groups that are stable under conditions for amide bond linking but can be easily cleaved without damaging the formed peptide chain.

[0159] 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.

[0160] 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.

[0161] Recombinant production Alternatively, the polypeptide domain of the gp130-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 secretory leader sequence (signal peptide) is incorporated into the polypeptide. The recombinant protein may be purified and concentrated for further use, including integration.

[0162] Synthesis of nucleic acid sequences encoding gp130 binding molecules In some embodiments, the polypeptide domain of the gp130-binding molecule is produced by a recombinant method using a nucleic acid sequence encoding the polypeptide domain of the gp130-binding molecule (or a fusion protein containing the polypeptide domain of the gp130-binding molecule). The nucleic acid sequence encoding the desired polypeptide domain of the gp130-binding molecule can be synthesized by chemical means using an oligonucleotide synthesizer.

[0163] 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 a gp130 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.

[0164] The nucleic acid molecules encoding the polypeptide domain (and its fusion) of the gp130 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).

[0165] The nucleic acid sequences encoding the polypeptide domain of the gp130-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 gp130-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.

[0166] Methods for constructing DNA sequences encoding the polypeptide domain of the gp130-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 gp130-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 gp130-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.

[0167] The polypeptide domain of the gp130-binding molecule of this disclosure may be produced not only directly by recombination, but also as a fusion polypeptide with a heterologous polypeptide, such as a signal sequence, or another polypeptide having a specific cleavage site at the N-terminus or C-terminus of the mature gp130-binding molecule. Generally, the signal sequence may be a component of the vector or part of a coding sequence inserted into the vector. The selected heterologous signal sequence is preferably one that is recognized and processed by a host cell (i.e., cleaved by a signal peptidase). In some embodiments, the signal sequence is a signal sequence that is naturally associated with the gp130-binding molecule (i.e., a human gp130 signal sequence). The incorporation of the signal sequence depends on whether it is desirable for the gp130-binding molecule to be secreted from the recombinant cell from which the gp130-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, the 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 gp130-binding molecules to be secreted extracellularly into the culture medium, as described in Singh, U.S. Patent No. 7,198,919B1.

[0168] If the polypeptide domain of the gp130-binding molecule to be expressed is to be expressed as a chimeric protein (e.g., a fusion protein containing the gp130-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 gp130-binding molecule and a second sequence encoding all or part of the heterologous polypeptide. For example, the polypeptide domain of the gp130-binding molecule described herein may be fused with a hexahistidine tag (SEQ ID NO: 281) to facilitate the purification of the protein expressed by bacteria, or with a hemagglutinin tag to facilitate the purification of the protein expressed in eukaryotic cells. The first and second should not be understood as limitations on the orientation of the elements of the fusion protein, as the heterologous polypeptide can be ligated to either the N-terminus and / or C-terminus of the polypeptide domain of the gp130-binding molecule. For example, the N-terminus may be ligated to a targeting domain, and the C-terminus may be ligated to a hexahistidine tag (SEQ ID NO: 281) purification handle.

[0169] A back-translated gene can be constructed using the complete amino acid sequence of the polypeptide domain of the gp130-binding molecule (or fusion / chimera) to be expressed. DNA oligomers containing the nucleotide sequence encoding the polypeptide domain of the gp130-binding molecule can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

[0170] In some embodiments, the nucleic acid sequence encoding the polypeptide domain of the gp130 binding molecule may be “codon-optimized” to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide variety of expression systems, including mammalian host cells, yeast host cells, and bacterial host cells, are well known in the art, and there are online tools for providing codon-optimized sequences for expression in various host cell types. For example, see Hawash, et al., (2017) 9:46-53, and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols See David Hacker (Human Press New York) for further information. Additionally, there are various web-based online software packages freely available to assist in the preparation of codon-optimized nucleic acid sequences.

[0171] Expression vector Once assembled (by synthesis, site-directed mutagenesis, or other means), the nucleic acid sequence encoding the polypeptide domain of the gp130-binding molecule is inserted into the 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.

[0172] 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.

[0173] The expression vectors for the polypeptide domain of the gp130-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 gp130-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 gp130 binding molecule, with respect to its potential secondary structure.

[0174] 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.

[0175] 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.

[0176] 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), heterozoaminal 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.

[0177] 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.

[0178] 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.

[0179] host cell Furthermore, this disclosure provides prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding the polypeptide domain of a gp130-binding molecule. The cells of this disclosure are transfected cells, i.e., cells into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding the polypeptide domain of a gp130-binding molecule, has been introduced by recombinant DNA. Progeny of such cells are also considered to be within the scope of this disclosure.

[0180] 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 gp130 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.

[0181] In some embodiments, the recombinant polypeptide domain of the gp130-binding molecule or its biologically active variant 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)).

[0182] 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.

[0183] The polypeptide domain of the gp130 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).

[0184] In some embodiments, the recombinant polypeptide domain of the gp130-binding molecule may or may not be glycosylated, depending on the host organism used to produce the gp130-binding molecule. When bacteria are selected as the host, the polypeptide domain of the produced gp130-binding molecule may be deglycosylated. On the other hand, eukaryotic cells may glycosylate the recombinant polypeptide domain of the gp130-binding molecule.

[0185] 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.).

[0186] Transfection The expression construct can be introduced into host cells to produce the recombinant polypeptide domain of the gp130-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.

[0187] 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).

[0188] 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.

[0189] Recombinant protein recovery If a secretion leader sequence is used, the recombination-produced gp130-conjugated polypeptide can be recovered from the culture medium as a secretion polypeptide. Alternatively, the gp130-conjugated 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.

[0190] 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.

[0191] The recombinant polypeptide domain of the gp130-binding molecule produced by the transformed host can be purified according to any suitable method. The gp130-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 gp130-binding molecule.

[0192] Recombinant polypeptides in substantially purified form can be used as therapeutic agents, for example, as described herein.

[0193] The biological activity of the recombinant polypeptide domain of the gp130-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 gp130 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).

[0194] How to use In some embodiments, compositions comprising gp130-binding molecules are useful in the treatment of human diseases, including autoimmune and inflammatory diseases, infections, and neoplasms. In one embodiment, the Disclosure provides a method for modulating the activity of gp130-expressing cells by subjecting them to a gp130-binding molecule in an amount sufficient to interfere with the activity of a gp130-containing receptor. Furthermore, the Disclosure provides a method for modulating the activity of gp130-expressing cells in a mixed cell population, comprising the step of contacting the cell population with the gp130-binding molecule or complex of the Disclosure in vivo and / or ex vivo in an amount sufficient to interfere with the activity of a gp130-containing receptor. In some embodiments, the gp130-binding molecule of the Disclosure is an inhibitor of the activity of a receptor (e.g., IL6) on which gp130 forms a subunit. gp130 forms a subunit of the IL6 receptor, and as previously discussed, IL6 inhibitors have demonstrated usefulness in the treatment of autoimmune and inflammatory diseases, infections, and neoplasms.

[0195] Autoimmune diseases and inflammatory diseases Disorders that are subject to treatment with the gp130-binding molecules of this disclosure (including pharmaceutically acceptable formulations comprising gp130-binding molecules and / or recombinant viruses encoding such gp130-binding molecules, or nucleic acid molecules encoding such gp130-binding molecules) include organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, and inflammatory bowel disease (IB). D) Crohn's disease, diabetes including type 1 or type 2 diabetes, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteroarthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative enthesopathy arthropathy syndrome) This includes inflammatory or autoimmune diseases, including but not limited to enthemismarthritis, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarthritis, polyarthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteritis arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (seronegative enthesopathy arthropathy syndrome).

[0196] Other examples of proliferative and / or differentiation disorders that are subject to treatment with the gp130-binding molecules of this disclosure (including pharmaceutically acceptable formulations containing gp130-binding molecules and / or nucleic acid molecules encoding such gp130-binding molecules, including recombinant viruses encoding such gp130-binding molecules) include, but are not limited to, skin disorders. Skin disorders may involve abnormal activity of cells or groups of cells or layers in the dermis, epidermis, or subcutaneous tissue layers, and may involve abnormalities at the dermal-epidermal junction. For example, skin disorders may involve abnormal activity of keratinocytes (e.g., hyperproliferative basal keratinocytes and keratinocytes just above the basal layer), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more of the epidermal layers, such as the basal layer (germinal layer), spinous layer, granular layer, clear layer, or stratum corneum. In other embodiments, the disorder may be accompanied by abnormal activity of dermal cells, such as dermal endothelium, fibroblasts, or immune cells (e.g., mast cells or macrophages) found in the dermal layer, such as the papillary layer or reticular layer.

[0197] Examples of inflammatory or autoimmune skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), such as exfoliative dermatitis or atopic dermatitis, keratosis pilaris, pityriasis rosacea, parapsoriasis, and lichenoid rash. This includes lichenoids, lichen planus, lichen pemphigoid, ichthyosis-like dermatosis, keratodermas, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, bullous pemphigoid (e.g., ocular scarring pemphigoid or bullous pemphigoid), urticaria, prokeratosis, rheumatoid arthritis with hyperproliferation and inflammation of epithelial-associated cells lining the joint capsule; dermatitis, e.g., seborrheic dermatitis and photodermatitis; keratosis, e.g., seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, and follicular keratosis; acne vulgaris; keloids and prevention of keloid formation; nevi; human papillomavirus (HPV) infections, including warts, condyloma or genital warts, and sexually transmitted warts; vitiligo; lichen planus; and keratitis. The skin disorder may be dermatitis, such as atopic dermatitis, allergic dermatitis, or psoriasis.

[0198] The compositions of this disclosure (including pharmaceutically acceptable formulations comprising a gp130-binding molecule and / or a recombinant virus encoding such a gp130-binding molecule, or a nucleic acid molecule encoding such a gp130-binding molecule) may also be administered to patients who have (or are likely to have) psoriasis or a psoriatic disorder. The term “psoriasis” is intended to have its medical meaning, namely, a disease affecting the skin and producing raised, thickened, scaly, or non-scaly lesions. The lesions are usually well-defined, erythematous papules covered with overlapping, shiny scales. The scales are typically silvery-white or slightly opalescent. Nail complications are frequent, resulting in hollowing, separation, thickening, and discoloration of the nails. Psoriasis can also be associated with arthritis, which can be severely detrimental. Keratinocyte hyperproliferation, along with epidermal inflammation and reduced keratinocyte differentiation, is a key feature of psoriatic epidermal hyperplasia. Several mechanisms have been proposed to explain the keratinocyte hyperproliferation characteristic of psoriasis. Impairment of cellular immunity has also been linked to the development of psoriasis. Examples of psoriatic disorders include chronic stationary psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, psoriasis vulgaris, eruptive psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or localized pustular psoriasis.

[0199] Combination with adjuvant therapy agents This disclosure provides the use of the gp130 conjugate molecule of this disclosure in combination with one or more additional activators ("adjuvants"). Such additional combinations are synonymous with "adjuvant combinations" or "adjuvant combination therapies," and therapeutic agents used in combination with the gp130 conjugate molecule of this disclosure are referred to as "adjuvants." As used herein, the term "adjuvants" includes agents that can be administered or introduced separately, for example, agents that can be formulated separately for separate administration (for example, as they may be provided in a kit), and / or therapies that can be administered or introduced in combination with the gp130 conjugate molecule.

[0200] As used herein, the term “in combination with” refers to the administration of a first agent and at least one further agent (i.e., a second, third, fourth, fifth, etc.) to a subject when used in relation to the administration of multiple agents to a subject. For the purposes of the present invention, an agent (e.g., a gp130 conjugate molecule) is considered to be administered in combination with a second agent if the biological effect resulting from the administration of the first agent persists to such an extent that the therapeutic effects of the first and second agents overlap when the second agent (e.g., an immune checkpoint pathway modulator) is administered. For example, PD1 immune checkpoint inhibitors (e.g., nivolumab or pembrolizumab) are typically administered by IV infusion every two or three weeks, whereas the gp130 conjugate molecules of this disclosure are typically administered more frequently, for example, daily, twice daily, or weekly. However, even if the first agent is administered at a considerable time (e.g., several days or weeks) after the administration of the second agent, the first agent (e.g., pembrolizumab) may provide a long-lasting therapeutic effect, and the second agent (e.g., a gp130 binding molecule) may provide a therapeutic effect for as long as the therapeutic effect of the first agent continues, so that the second agent is considered to have been administered in combination with the first agent. In one embodiment, an agent is considered to have been administered in combination with the second agent if the first and second agents are administered simultaneously (within 30 minutes of each other), concurrently, or consecutively. In some embodiments, the first agent is considered to have been administered "concurrently" with the second agent if the first and second agents are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other. The term “in combination with” is also understood to apply to situations in which a first agent and a second agent are co-prescribed in a single pharmaceutically acceptable formulation, and the co-formulation is administered to the subject. In certain embodiments, for example, when an agent is administered before one or more other agents, the gp130 conjugate molecule and the adjuvant are administered or applied sequentially. In other embodiments, for example, when two or more agents are administered simultaneously or nearly simultaneously, the gp130 conjugate molecule and the adjuvant are administered simultaneously.Two or more agents may be present in two or more separate formulations, or they may be combined to form a single formulation (i.e., a co-formulation). Whether the agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0201] A useful adjuvant in the treatment of inflammatory or autoimmune disorders. In some embodiments, the method further includes administering the gp130-binding molecule of the Disclosure in combination with one or more adjuvants selected from the group consisting of corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTor inhibitors, IMDH inhibitors, biologics, vaccines, and therapeutic antibodies. In certain embodiments, the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BlyS, CD11a, CD20, CD25, CD3, CD52, IgE, IL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

[0202] In some embodiments, the adjuvant is one or more agents selected from the group consisting of corticosteroids (including, but not limited to, prednisone, budesonide, and prednirisone), Janus kinase inhibitors (including, but not limited to, tofacitinib (Xeljanz®)), calcineurin inhibitors (including, but not limited to, cyclosporine and tacrolimus), mTor inhibitors (including, but not limited to, sirolimus and everolimus), IMDH inhibitors (including, but not limited to, azathioprine, leflunomide, and mycophenolic acid), biologics such as abatacept (Orencia®) or etanercept (Enbrel®), and therapeutic antibodies.

[0203] Examples of therapeutic antibodies that may be administered as adjuvants in combination with the gp130-binding molecule of this disclosure in the treatment of autoimmune diseases include anti-CD25 antibodies (e.g., daclizumab and basiliximab), anti-VLA-4 antibodies (e.g., natalizumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-CD20 antibodies (e.g., rituximab, ocrelizumab), anti-TNF antibodies (e.g., infliximab and adalimumab), anti-IL6R antibodies (e.g., tocilizumab), anti-TNFα antibodies (e.g., adalimumab (Humira®), golimumab, and infliximab), This includes, but is not limited to, liximab, anti-integrin-α4β7 antibodies (e.g., vedolizumab), anti-IL17a antibodies (e.g., brodalumab or secukinumab), anti-IL4Rα antibodies (e.g., dupilumab), anti-RANKL antibodies, IL6R antibodies, anti-IL1β antibodies (e.g., canakinumab), anti-CD11a antibodies (e.g., efalizumab), anti-CD3 antibodies (e.g., muramonab), anti-IL5 antibodies (e.g., mepolizumab, reslizumab), anti-BLyS antibodies (e.g., belimumab), and anti-IL12 / IL23 antibodies (e.g., ustekinumab).

[0204] Many therapeutic antibodies have been approved for clinical use for autoimmune diseases. Examples of antibodies approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of autoimmune diseases in subjects suffering from autoimmune diseases are shown in the table below, which may be administered as adjuncts in combination with the gp130 conjugate molecules of this disclosure (and optionally, further adjuncts) for the treatment of indicated autoimmune diseases.

[0205] (Table 4) Antibodies useful as adjuvants in the treatment of autoimmune and inflammatory diseases TIFF2026063000000037.tif149160

[0206] The aforementioned antibodies of Table 4 useful as adjuvants in the practice of the methods of the present disclosure may be administered alone, or in the form of any antibody-drug conjugate (ADC) comprising an antibody, a linker, and one or more drugs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 drugs), or may be administered in a modified form (e.g., PEGylated).

[0207] Treatment of neoplastic diseases The present disclosure provides a method of using a gp130-binding molecule (or a nucleic acid encoding a gp130-binding molecule, including a recombinant vector encoding the gp130-binding molecule, as well as eukaryotic and prokaryotic cells modified to express the gp130-binding molecule) as described herein in the treatment of a subject afflicted with a neoplastic disease disorder or condition by administration of a therapeutically effective amount thereof.

[0208] Neoplasms to be treated: The compositions and methods of the present disclosure are useful in the treatment of neoplastic diseases characterized by the presence of neoplasms, including benign and malignant neoplasms, and in the treatment of subjects afflicted with neoplastic diseases.

[0209] Examples of benign neoplasms that may be treated using the compositions and methods of the present disclosure include, but are not limited to, adenomas, fibromas, hemangiomas, and lipomas. Examples of premalignant neoplasms that may be treated using the compositions and methods of the present disclosure include, but are not limited to, hyperplasia, dysplasia, metaplasia, and atypia. Examples of malignant neoplasms that may be treated using the compositions and methods of the present disclosure typically include carcinomas (cancers arising from epithelial tissues such as the skin or tissues lining the viscera), leukemias, lymphomas, and sarcomas, which are typically derived from bone, fat, muscle, blood vessels, or connective tissue, but are not limited thereto. The term neoplasm also includes virus-induced neoplasms, such as warts, and EBV-induced diseases (i.e., infectious mononucleosis), scar formation, proliferative vascular diseases including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion.

[0210] The term “neoplasm” includes breast cancer; sarcomas (including, but not limited to, osteosarcoma, angiosarcoma, and fibrosarcoma), leukemia, lymphoma, genitourinary cancers (including, but not limited to, ovarian cancer, urethral cancer, bladder cancer, and prostate cancer); gastrointestinal cancers (including, but not limited to, colon cancer, esophageal cancer, and stomach cancer); lung cancer; myeloma; pancreatic cancer; liver cancer; kidney cancer; endocrine cancer; skin cancer; and tumors of the brain or central and peripheral nervous system (CNS), malignant or benign, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders; cervical intraepithelial neoplasia; intestinal polyps; oral leukoplakia; histiocytosis, hyperproliferative scars including keloids, hemangiomas; hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis; hyperkeratosis, and papular scaly rashes including arthritis.

[0211] The term neoplasm includes carcinoma. The term "carcinoma" refers to malignant tumors of epithelial or endocrine tissue, including respiratory carcinomas, gastrointestinal carcinomas, genitourinary carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine carcinomas, and melanomas. The term neoplasm includes adenocarcinoma. "Adenocarcinoma" refers to carcinomas that originate from glandular tissue, or carcinomas in which tumor cells form recognizable glandular structures.

[0212] As used herein, the term “hematopoietic neoplasm” refers to a neoplasmic disease involving hyperplastic / neoplastic cells arising from hematopoietic origin, such as myelocytes, lymphocytes, or erythrocytes, or their progenitor cells.

[0213] Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myelocytic and lymphocytic disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related progenitor neoplasms, and unclear lineages of acute leukemia. Exemplary myelopathies that are subject to treatment in accordance with this disclosure include, but are not limited to, acute promyeloid leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).

[0214] Lymphoid neoplasms include, but are not limited to, progenitor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin lymphoma, and immunodeficiency-associated lymphoproliferative disorders. Exemplary lymphoid disorders that are subject to treatment in accordance with this disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-series ALL and T-series ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), pilocytic cell leukemia (HLL), and Waldenström macroglobulinemia (WM).

[0215] In some cases, hematopoietic neoplasms are caused by poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term “hematopoietic neoplasm” refers to malignant lymphomas, including but not limited to non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin’s disease, and Reed-Sternberg disease.

[0216] The determination of whether a subject has a "neoplasmic disease" refers to a physician's decision regarding a subject based on accepted, available information in the field for identifying a disease, disorder, or condition, including but not limited to X-rays, CT scans, conventional clinical diagnostic tests (e.g., blood cell counts), genomic data, protein expression data, and immunohistochemistry, which determine whether the subject requires or would benefit from treatment.

[0217] Combination of gp130 binding molecule and antineoplastic adjuvant: This disclosure provides the use of the gp130 conjugate molecule of this disclosure in combination with one or more additional active antineoplastic agents ("adjuvants") for treating neoplastic diseases. Such additional combinations are synonymous with "antineoplastic adjuvant combinations" or "antineoplastic adjuvant combination therapies," and therapeutic agents used in combination with the gp130 conjugate molecule of this disclosure are referred to as "antineoplastic adjuvants." As used herein, the term "antineoplastic adjuvants" includes antineoplastic agents that can be administered or introduced separately, for example, those that can be formulated separately for separate administration (for example, as they may be provided in a kit), and / or therapies that can be administered or introduced in combination with the gp130 conjugate molecule.

[0218] Chemotherapy agents: In some embodiments, the antineoplastic adjuvant is a chemotherapeutic agent. In some embodiments, the adjuvant is a “cocktail” of several chemotherapeutic agents. In some embodiments, the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e.g., radiotherapy). The term “chemotherapeutic agent” includes alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines, and methylamelamamines including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamamine. Nitrogen mustards, e.g., thiorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembicin, fenestrinn, prednimustine, trophosphamide, uracil mustard; Nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics, e.g., acrasinomycin, a Cutinomycin, Ausuramycin, Azaserin, Bleomycin, e.g., Bleomycin A2, Cactinomycin, Calitiamycin, Carabicin, Caminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin and derivatives, e.g., Demethoxy-Daunomycin, 11-Deoxydaunorubicin, 13-Deoxydaunorubicin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, Doxorubicin, Epirubicin, E Sorbicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, N-methylmitomycin C; mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, yubenimex, dinostatin, solubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, dideazatetrahydrofolate, and folic acid; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testo Lactones; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofhamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisantren; edatrexate; defofamine; demecoltin; diazion; elformitin; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; podophyllin Citric acid; 2-ethylhydrazide; procarbazine; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipoproman; gasitosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel, nab-paclitaxel, and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate Platinum and platinum-coordinate complexes, e.g., cisplatin, oxaplatin, and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; taxanes, e.g., paclitaxel, docetaxel, cabazitaxel;This includes, but is not limited to, carminomycin, adriamycin, e.g., 4'-epiaadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; cortisin, and any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0219] The term “chemotherapeutic agent” also includes anti-hormone agents that work to modulate or inhibit hormonal effects on tumors, such as anti-estrogens including tamoxifen, raloxifen, the aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, onapristone, and toremifene, as well as anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0220] In some embodiments, antineoplastic adjuvants include cytokines or cytokine antagonists, e.g., IL-12, INFα, or anti-epidermal growth factor receptors, irinotecan; tetrahydrofolate antagonists, e.g., pemetrexed; antibodies against tumor antigens, monoclonal antibody-toxin complexes, T-cell adjuvants, bone marrow transplants, or antigen-presenting cells (e.g., dendritic cell therapy); antitumor vaccines; replicating viruses; signaling inhibitors (e.g., Gleevec® or Herceptin®); or immunomodulators to achieve additive or synergistic inhibition of tumor growth, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicad) This includes, but is not limited to, one or more chemical or biological agents found useful in the art for treating neoplasms, including, but not limited to, e(registered trademark) and Enbrel(registered trademark), interferon-β1a (Avonex(registered trademark)), and interferon-β1b (Betaseron(registered trademark)), as well as one or more of the aforementioned combinations as implemented in known chemotherapy regimens, including, but not limited to, TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily understood by those skilled in the art.

[0221] In some embodiments, the gp130-binding molecule is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor such as sunitinib, a PARP inhibitor such as olaparib, an EGFR inhibitor such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor such as epacadostat, and a tumor regression virus such as tarimozine-laharpalepbec (T-VEC).

[0222] Antitumor antigen antibody therapy as an adjunct agent In some embodiments, “antineoplastic adjuvants” include, but are not limited to, bispecific T-cell engagers (BITEs), biaffinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs, as therapeutic antibodies (including bispecific and trispecific antibodies) that bind to one or more tumor-associated antigens.

[0223] In some embodiments, the therapeutic antibody is HER2 (e.g., trastuzumab, pertuzumab, adtrastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilumumab), CD22 (e.g., moxetumomab pasudotox), CCR4 (e.g., magumizumab), IL23p19 (e.g., tildrakizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab), CD 38 (e.g., daratumumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate-binding protein, GD2 (e.g., dinuntuximab), GD3, IL6 (e.g., siltuximab), GM2, Le y The antibody is an antibody that binds to at least one tumor antigen selected from the group consisting of VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRa (e.g., oraltumumab), EGFR (e.g., cetuximab, panitumumab, and nesitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKLRAP, tenascin, integrin αVβ3, and integrin α4β1.

[0224] In some embodiments, therapeutic antibodies are immune checkpoint modulators for treating and / or preventing neoplasms in a subject, as well as diseases, disorders, or conditions associated with neoplasms. The term “immune checkpoint pathway” refers to a biological response induced by stimulating (e.g., upregulation of T cell activity) or inhibiting (e.g., downregulation of T cell activity) an immune response when a first molecule expressed on antigen-presenting cells (APCs) (e.g., a protein such as PD1) binds to a second molecule expressed on immune cells (e.g., T cells) (e.g., a protein such as PDL1), thereby modulating the immune response. The molecules involved in forming binding pairs that modulate the immune response are generally called “immune checkpoints.” In one embodiment, an immune checkpoint pathway modulator is a negative immune checkpoint pathway antagonist ("PD1 pathway inhibitor") that inhibits the binding of PD1 to PDL1 and / or PDL2. The term PD1 pathway inhibitor includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Examples of commercially available PD1 pathway inhibitors useful as adjuncts in the treatment of neoplasms include nivolumab (Opdivo®, BMS-936558, MDX1106, marketed by BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, marketed by Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco). This includes antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2, including but not limited to CA.Further PD1 pathway inhibitor antibodies, including but not limited to durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers Squibb), avelumab (MSB0010718C, Merck Serono / Pfizer), and SHR-1210 (Incyte), are in clinical development. Further antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149 (Genentech, Inc.), issued July 10, 2012; U.S. Patent No. 8,168,757 (Merck Sharp and Dohme Corp.), issued May 1, 2012; U.S. Patent No. 8,008,449 (Medarex), issued August 30, 2011; and U.S. Patent No. 7,943,743 (Medarex, Inc.), issued May 17, 2011.

[0225] Examples of antibody therapies approved by the FDA and usable as adjuvants for the treatment of neoplasms include atezolizumab, olaratumab, ixekizumab, trastuzumab, infliximab, rituximab, edrecolomab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, pertuzumab, brentuximab vedotin, ipilimumab, ofatumumab, certolizumab pegol, catumakisomab, and panitumumab. This includes bevacizumab, ramucirumab, siltuximab, enfortumab vetotin, polatuzumab vetotin, [fam]-trastuzumab deruxtecan, semiprimab, moxetumomab pasudotox, mogamuizumab, tildrakizumab, ibalizumab, durvalumab, inotuzumab, ozogamicin, avelumab, obinutuzumab, adtrastuzumab emtansine, cetuximab, tositumomab-I131, ibritumomab tiuxetan, gemtuzumab, and ozogamicin.

[0226] physical method In some embodiments, the antineoplastic adjuvant is one or more non-pharmacological modalities (e.g., local or total radiotherapy or surgery). As an example, the disclosure intends a treatment regime in which treatment is performed with a treatment regimen comprising a gp130 conjugate molecule and one or more antineoplastic adjuvants before or after a radiation stage. In some embodiments, the disclosure further intends the use of the gp130 conjugate molecule in combination with surgery (e.g., tumor resection). In some embodiments, the disclosure further intends the use of the gp130 conjugate molecule in combination with bone marrow transplantation, peripheral blood stem cell transplantation or other types of transplantation therapy.

[0227] In some embodiments, the methods of the present disclosure may include a combination of a gp130-binding molecule and the administration of an adjuvant in the form of cell therapy for treating neoplasms, autoimmune diseases, or inflammatory diseases. Examples of cell therapies intended for use in combination with the methods of the present disclosure include, but are not limited to, engineered T cell products containing one or more types of activated CAR-T cells, engineered TCR cells, tumor-infiltrating lymphocytes (TILs), and engineered Treg cells.

[0228] CARs useful in carrying out the present invention are prepared according to principles well known in the art. For example, see Eshhaar et al., U.S. Patent No. 7,741,465 B1, issued June 22, 2010; Sadelain, et al (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products include axicaptagensilolucel (marketed as Yescarta® by Gilead Pharmaceuticals) and tisagenlecleucel (marketed as Kymriah® by Novartis). In some embodiments, the CAR-T has a CAR selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3R□2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP, which specifically binds to cell surface molecules associated with tumor cells.

[0229] formulation Furthermore, the present disclosure provides a pharmaceutically acceptable formulation of the gp130 binding molecule of the present disclosure. Preferred formulations are determined by the intended method of administration and therapeutic use. The pharmaceutical dosage forms of the gp130 binding molecules described herein are essentially non-toxic and contain physiologically acceptable carriers that are not therapeutically useful. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and PEG. Carriers for topical or gel-based forms of polypeptides include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0230] Pharmaceutical compositions may also include pharmaceutically acceptable, non-toxic carriers, excipients, stabilizers, or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. Diluents are selected so as not to affect the biological activity of the combination. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecydimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g. The following are included: serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0231] Formulations used for in vivo administration are typically sterile. The compositions of this disclosure can be readily sterilized by filtration through a sterile filtration membrane.

[0232] Typically, the compositions are prepared as injectable solutions, either liquid solutions or suspensions. Solid forms suitable for dissolving or suspending in a liquid vehicle before injection can also be prepared. Preparations may be emulsified and, as described above, encapsulated in liposomes or microparticles, such as polylactides, polyglycolides, or copolymers to enhance the adjuvant effect (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The agents of this disclosure may be administered in the form of depot injections or graft preparations that can be formulated to release the active ingredient in a sustained or pulsatile manner. Pharmaceutical compositions are generally sterile, substantially isotonic, and fully comply with all Good Manufacturing Practices (GMP) of the U.S. Food and Drug Administration.

[0233] Vector delivery of polypeptide gp130 binding molecules In embodiments where the gp130-binding molecule is a polypeptide, such gp130-binding molecule may also be delivered to the target by administering a recombinant vector containing a nucleic acid sequence encoding the peptidyl gp130-binding molecule, which is functionally linked to an expression regulatory sequence in cells of the target tissue.

[0234] The expression vector may be a viral vector or a non-viral vector. The term "non-viral vector" refers to a self-replicating extrachromosomal circular DNA molecule that differs from the normal genome and is unnecessary for cell survival under non-selective conditions in which it can express a coding sequence in the target cell. Plasmids are an example of a non-viral vector. To facilitate transfection of target cells, target cells may be directly exposed with the non-viral vector under conditions that facilitate its uptake. 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).

[0235] In one embodiment, the nonviral vector may be provided in a nonviral delivery system. The nonviral delivery system is typically a complex that facilitates the transduction of a nucleic acid cargo into target cells, in which case the nucleic acid is complexed with an agent, such as a cationic lipid (DOTAP, DOTMA), a surfactant, a biologic (gelatin, chitosan), a metal (gold, magnetite), and a synthetic polymer (PLG, PEI, PAMAM). Lipid vector systems (Lee et al. (1997) Crit Rev Ther Drug Carrier Syst. 14:173-206); Polymer-coated liposomes (Marin et al., U.S. Patent No. 5,213,804, issued May 25, 1993; Woodle, et al., U.S. Patent No. 5,013,556, issued May 7, 1991); Cationic liposomes (Epand et al., U.S. Patent No. 5,283,185, issued February 1, 1994; Jessee, JA, U.S. Patent No. 5,578,475, issued November 26, 1996; Rose et al., U.S. Patent No. 5,279,833, issued January 18, 1994; Gebeyehu et al., issued August 2, 1994) A great many non-viral delivery systems are well known in the art, including U.S. Patent No. 5,334,761.

[0236] In another embodiment, the expression vector may be a viral vector. As used herein, the term viral vector is used in the conventional sense to refer to any obligate intracellular parasite that lacks both protein synthesis and energy generation mechanisms, and usually refers to any enveloped or non-enveloped animal virus commonly used to deliver exogenous transgenes to mammalian cells. Viral vectors may be replicable (e.g., substantially wild-type), conditionally replicable (engineered by recombination to replicate under certain conditions), or replication-deficient (substantially incapable of replicating the virus's lost function in the absence of a cell line capable of compensating for the virus's lost function). Viral vectors may have certain modifications to "replicate specifically," i.e., preferentially replicate in a particular cell type or phenotypic state, e.g., cancer. Viral vector systems useful in the implementation of the gp130 conjugate molecule include, for example, native viral vector systems or recombinant viral vector systems. Examples of viruses useful in the implementation of the gp130 conjugate molecule include recombinantly modified enveloped or non-enveloped DNA viruses and RNA viruses. For example, viral vectors may be derived from the genomes of human or bovine adenoviruses, vaccinia viruses, lentiviruses, herpesviruses, adeno-associated viruses, human immunodeficiency viruses, Sindbisviruses, and retroviruses (including, but not limited to, Roussarcoma virus), as well as hepatitis B virus. Typically, the gene of interest is inserted into such a vector, usually along with an associated viral genome sequence, to enable the packaging of a gene construct, and the vector is then used to infect susceptible host cells to express the gene of interest (e.g., a target antigen).

[0237] The expression vector may encode one or more polypeptides in addition to the target antigen. When multiple polypeptides are expressed, as in the implementation of this gp130 conjugate molecule, each polypeptide may be functionally linked to an expression regulatory sequence (monocistronic), or the multiple polypeptides may be encoded by a polycistronic construct in which the multiple polypeptides are expressed under the control of a single expression regulatory sequence. In one embodiment, the expression vector encoding the target antigen may optionally further encode one or more immunological modulators. Examples of immunological modulators useful in the implementation of this gp130 conjugate molecule include, but are not limited to, cytokines. Examples of such cytokines include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-12, TNF-α, interferon-α, interferon-α-2b, interferon-β, interferon-γ, GM-CSF, MIP1-α, MIP1-β, MIP3-α, TGF-β, and one or more other suitable cytokines that can modulate the immune response, such as interleukins. The expressed cytokines may be directed for intracellular expression, or they may be expressed together with signal sequences for extracellular presentation or secretion.

[0238] The expression vector may optionally provide a further expression cassette containing a nucleic acid sequence encoding a “rescue” gene. The “rescue gene” is a nucleic acid sequence that, when expressed, causes cells to become susceptible to death by extrinsic factors or induces an intracellular toxic state that leads to cell death. The presence of the rescue gene enables selective cell death of transduced cells. Therefore, when the construct is incorporated into cells of a mammalian target, the rescue gene provides an additional safety measure to prevent the effects of a vector system that has the undesirable propagation or replication ability of transduced cells. In one embodiment, the rescue gene is a thymidine kinase (TK) gene (see, for example, Woo, et al., U.S. Patent No. 5,631,236, issued May 20, 1997, and Freeman, et al., U.S. Patent No. 5,601,818, issued February 11, 1997), and cells expressing the TK gene product are more susceptible to selective cell death by ganciclovir administration.

[0239] Dosage Furthermore, this disclosure provides the administration of a recombinant vector or cells comprising a nucleic acid sequence encoding a polypeptide gp130-binding molecule or polypeptide gp130-binding molecule in a therapeutically or prophylactically effective dose to subjects suffering from a disease, disorder, or condition, or subjects at risk of developing such a disease, disorder, or condition. The dose of the pharmaceutical composition comprising the gp130-binding molecule, vector, or cells depends on factors such as the route of administration, the disease to be treated, and the physical characteristics of the subject, including age, weight, and overall health. Typically, the amount of gp130-binding molecule contained in a single dose may be an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity. The pharmaceutical compositions disclosed herein are available in concentrations of 0.01 to 500 mg / kg (e.g., 0.01 to 450 mg, 0.01 to 400 mg, 0.01 to 350 mg, 0.01 to 300 mg, 0.01 to 250 mg, 0.01 to 200 mg, 0.01 to 150 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 10 mg, 0.01 to 1 mg, 0.). 500mg / kg, 1~500mg / kg, 5~500mg / kg, 10~500mg / kg, 50~500mg / kg, 100~500mg / kg, 150~500m g / kg, 200~500mg / kg, 250~500mg / kg, 300~500mg / kg, 350~500mg / kg, 400~500mg / kg, or 450 The gp130 binding molecule described herein may be present in doses of approximately 1 to approximately 100 mg / kg (e.g., approximately 1 to approximately 90 mg / kg, approximately 1 to approximately 80 mg / kg, approximately 1 to approximately 70 mg / kg, approximately 1 to approximately 60 mg / kg, approximately 1 to approximately 50 mg / kg, approximately 1 to approximately 40 mg / kg, approximately 1 to approximately 30 mg / kg, approximately 1 to approximately 20 mg / kg, approximately 1 to approximately 10 mg / kg, approximately 10 to approximately 100 mg / kg, approximately 20 to approximately 100 mg / kg, approximately 30 to approximately 100 mg / kg, approximately 40 to approximately 100 mg / kg, approximately 50 to approximately 100 mg / kg, approximately 60 to approximately 100 mg / kg, approximately 70 to approximately 100 mg / kg, approximately 80 to approximately 100 mg / kg, or approximately 90 to approximately 100 mg / kg).In some embodiments, the pharmaceutical compositions of this disclosure may contain the binding proteins described herein in doses of 0.01 to 20 mg / kg (e.g., 0.01 to 15 mg / kg, 0.01 to 10 mg / kg, 0.01 to 8 mg / kg, 0.01 to 6 mg / kg, 0.01 to 4 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg, 0.01 to 0.05 mg / kg, 0.05 to 20 mg / kg, 0.1 to 20 mg / kg, 1 to 20 mg / kg, 2 to 20 mg / kg, 4 to 20 mg / kg, 6 to 20 mg / kg, 8 to 20 mg / kg, 10 to 20 mg / kg, 15 to 20 mg / kg). The physician may adjust the dose according to conventional factors such as the severity of the disease and various parameters of the subject.

[0240] Pharmaceutical compositions containing the gp130 binding molecule described herein can be administered to subjects requiring it, for example, once or multiple times (e.g., 1 to 10 times or more) daily, weekly, monthly, twice a year, annually, or as medically required. Dosages may be provided in a single or multiple dosing plan. The interval between dosing may be reduced when the medical condition improves, and increased when the patient's health deteriorates. The course of therapy may be a single dose or multiple doses over a period of time. In some embodiments, a single dose is used. In some embodiments, two or more divided doses are used, administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. Each dose administered in such a divided dose protocol may be the same or different. A multi-day dosing protocol may be provided over a period of time, taking into account the subject's response to the treatment, including side effects, and the adjustment of side effects as discussed above, by monitoring the administration by a person skilled in the art (e.g., a physician).

[0241] For preventive purposes, pharmaceutical compositions or medical agents are administered to patients who are susceptible to the disease or otherwise at risk of the disease, in amounts sufficient to eliminate the disease, reduce the risk of the disease, lessen the severity of the disease, or delay the onset of the disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes exhibited during the development of the disease.

[0242] In some embodiments, the condition to be treated is a chronic condition (e.g., a chronic infection, i.e., an infection that is not cleared by the host immune system within a period of up to one week, two weeks, etc.). In some cases, the chronic condition involves the integration of pathogenic genetic elements into the host genome, e.g., retroviruses, lentiviruses, hepatitis B virus, etc. In other cases, chronic infections, e.g., certain intracellular bacterial or protozoan pathogens, are caused by pathogenic cells within host cells. Furthermore, in some embodiments, infections, like herpesviruses or human papillomaviruses, have an incubation period. In such cases, the course of therapy may involve long-term administration of gp130-binding molecules, including continuous administration in the substantial absence of chronic condition symptoms to prevent recurrence of the chronic condition or its symptoms.

[0243] For preventative purposes, relatively small doses may be administered over a long period at relatively infrequent intervals. Some patients may continue to receive treatment. For other therapeutic purposes, relatively large doses may be required at relatively short intervals until the progression of the disease slows down or ends, preferably until the patient shows partial or complete remission of disease symptoms. After that, this patent can be administered in a preventative manner.

[0244] Route of administration The administration of the gp130-binding molecule described herein can be achieved by any method 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, intranodal injection, transdermal delivery, transmucosal delivery, iontophoretic delivery, intralymphatic injection (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), respiratory inhaler including nebulizer, intraocular injection, intraperitoneal injection, intrafocal injection, intraovarian injection, intracerebral or intracerebral injection, intraventricular injection (ICVI), and various other methods recognized in the art. Administration to the subject may be achieved by intravenous administration, as a bolus, or by continuous infusion over a period of time. Examples of parenteral administration routes include, for example, intravenous, intradermal, subcutaneous, transdermal (local), transmucosal, and rectal administration. The gp130 conjugate molecule may be administered as a single dose, or continuously, for example, by a continuous pump, or at periodic intervals (daily, bi-weekly, monthly) over a period of time that may occur for one week, two weeks, one month, two months, three months, or longer. The desired time intervals between multiple doses of the gp130 conjugate molecule can be determined by those skilled in the art.

[0245] As discussed above, the compositions of this disclosure may be used in combination with one or more further therapeutically active agents. As used herein, the term “in combination with” refers to the administration of a first agent and at least one further (i.e., second, third, fourth, fifth, etc.) adjutant to a subject, when used in relation to the administration of multiple agents to a subject. For the purposes of the present invention, an agent (e.g., a gp130 binding molecule) is considered to be administered in combination with an adjutant if the biological effect resulting from the administration of the first agent persists in the subject at the time of administration of the adjutant in such a way that the therapeutic effects of the first and second agents overlap. Even if the first agent is administered at a time considerably far removed from the time of administration of the adjutant (e.g., several days or weeks), the administration of the first agent may yield a therapeutic effect over a long period, and the administration of the adjutant may yield its therapeutic effect while the therapeutic effect of the first agent persists in such a way that the adjutant is considered to have been administered in combination with the first agent. In one embodiment, an agent is considered to be administered in combination with an adjuvant if the first agent and the second agent are administered simultaneously (within 30 minutes of each other), concurrently, or consecutively. In some embodiments, the first agent is considered to be administered "contemporarily" with the adjuvant if the first agent and the adjuvant are administered within approximately 24 hours of each other, preferably within approximately 12 hours of each other, preferably within approximately 6 hours of each other, preferably within approximately 2 hours of each other, or preferably within approximately 30 minutes of each other. The term "in combination with" is also understood to apply to situations in which the first agent and the adjuvant are co-formulated in a single pharmaceutically acceptable formulation and the co-formulation is administered to the subject. In certain embodiments, for example, if an agent is administered before one or more other agents, the first agent and the adjuvant are administered or applied consecutively. In other embodiments, the first agent and the adjuvant are administered simultaneously. For example, when two or more drugs are administered simultaneously or nearly simultaneously, the two or more drugs may be present in two or more separate formulations, or they may be combined to form a single formulation (i.e., a co-formulation). Regardless of whether the drugs are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0246] kit This disclosure also intends kits comprising pharmaceutical compositions of gp130 conjugate molecules. In some embodiments, the kit further comprises an auxiliary pharmaceutical composition comprising adjuvants such as those discussed above for use in combination therapy with gp130 conjugate molecules. The kit generally takes the form of a physical structure containing various components as described below, and can be used, for example, in the implementation of the above methods. The kit may contain gp130 conjugate molecules in the form of a pharmaceutical composition suitable for administration to a subject ready for use, or it may contain gp130 conjugate molecules in a form that requires preparation, e.g., thawing, reconstitution, or dilution before administration. If the gp130 conjugate molecules are in a form that requires reconstitution by the user, the kit may also include a sterile container providing a reconstitution medium, such as a buffer or pharmaceutically acceptable excipient. 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 kit may further include a label or accompanying document containing identifying information of the components contained in the kit, and instructions for use. Each component of the kit may be sealed in an individual container, or all the various containers may be in a single packaging container. The label or accompanying information may contain manufacturer information such as lot number and expiration date. The label or accompanying information may be, for example, integrated into the surface of the physical structure containing the components, placed separately within the physical structure, or affixed to the components of the kit (e.g., ampoules, syringes, or vials). The label or accompanying information may be provided in physical form or on a computer-readable medium. In some embodiments, the actual instructions are not present in the kit, and the kit provides means for obtaining the instructions from a remote source, for example, via an internet site, including obtaining the instructions by secure access through the provision of a password (or a scannable code such as a barcode or QR code on the surface of the gp130 conjugated molecule container or kit containing it), in accordance with government regulations (e.g., HIPAA). [Examples]

[0247] The following examples are provided to fully disclose and illustrate to those skilled in the art how to prepare and use the gp130-binding molecules, and are not intended to limit the scope of what the inventors consider to be gp130-binding molecules, 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, gp130-binding molecules 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.

[0248] 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.

[0249] Example 1. Immunization Protocol The process for isolating anti-hgp130 VHH was initiated by immunizing camels with polypeptides corresponding to amino acids 23-619 of hgp130 (UNIPROT reference number P40189). The process for isolating anti-mgp130 VHH was initiated by immunizing camels with the 201 amino acid extracellular domain of mgp130 and amino acids 23-617 of the mgp130 precursor (UNIPROT reference number Q00560). For each antigen, VHH was identified and isolated using the following methodology.

[0250] 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.

[0251] 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 VHH-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.

[0252] Example 3: Isolation of antigen-specific VHH Biopanning of phage libraries was performed to identify VHHs that bind to IFNgR1. 96-well plates were coated with IFNgR1, and phage libraries were incubated in each well to allow phage-expressed IFNgR1-reactive VHHs to bind to the IFNgR1 on the plate. Non-specifically bound phages were washed away, and specifically bound phages were isolated. After selection, the enriched phage libraries expressing IFNgR1-reactive VHHs were amplified in TG1 cells. The biopanning process described above was repeated 2-3 times to enrich the library for IFNgR1-reactive VHHs.

[0253] 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 IFNgR1-coated plates to identify positive VHH conjugates that selectively bound to IFNgR1. The 96-well plates were coated with IFNgR1 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 wells was at least 3 times that of the PBS-coated control were considered to provide specific binding to IFNgR1. Positive clones were sequenced, and their sequences were analyzed to identify unique chronotypes.

[0254] Example 5. Evaluation of coupling affinity via surface plasmon resonance To evaluate binding via SPR as described below, representative examples were selected from each hgp130 VHH and mgp130 VHH chronotype prepared according to Examples 1-3. The binding affinity of hgp130 binding molecules corresponding to SEQ ID NOs 2, 3, 4, 5, 6, and 7 was evaluated using surface plasmon resonance (SPR) in general the following manner. 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 ligands were 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 IL2Rb 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.

[0255] 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.

[0256] Sequence information SEQUENCE LISTING <110> SYNTHEKINE, INC. <120> GP130 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> 288 <170> PatentIn version 3.5 <210> 1 <211> 918 <212> PRT <213> Homo sapiens <400> 1 Met Leu Thr Leu Gln Thr Trp Leu Val Gln Ala Leu Phe Ile Phe Leu 1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30 Pro Glu Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55 60 Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr Thr 65 70 75 80 Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85 90 95 Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu 100 105 110 Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys 115 120 125 Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205 Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220 Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu 225 230 235 240 Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys 245 250 255 Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile 260 265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile 305 310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335 Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360 365 Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala 370 375 380 Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu 385 390 395 400 Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415 Pro Ala Cys Asp Phe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425 430 Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445 Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr 465 470 475 480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485 490 495 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505 510 Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu Pro Val 530 535 540 Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr 545 550 555 560 Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 605 Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro 610 615 620 Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys 625 630 635 640 Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655 Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670 Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685 Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700 Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn 705 710 715 720 Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725 730 735 Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 740 745 750 Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765 His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp 785 790 795 800 His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys 805 810 815 Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830 Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845 Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 850 855 860 Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875 880 Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895 Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910 Gly Gly Tyr Met Pro Gln 915 <210> 2 <211> 129 <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 Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ala Ile Ala Ser Gly Tyr 20 25 30 Ile Asp Ser Arg Trp Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45 Glu Arg Glu Gly Val Ala Ala Ile Trp Pro Gly Gly Gly Leu Thr Val 50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ala 65 70 75 80 Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr 85 90 95 Ala Met Tyr Tyr Cys Ala Ala Gly Ser Pro Arg Met Cys Pro Ser Leu 100 105 110 Glu Phe Gly Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <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 Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Pro Gly Phe Thr Ser Asn Ser Cys 20 25 30 Gly Met Asp Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ser Ile Ser Thr Asp Gly Thr Thr Gly Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Lys Ala Lys Asp Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Met Tyr Ser Cys Lys 85 90 95 Thr Lys Asp Gly Thr Ile Ala Thr Met Glu Leu Cys Asp Phe Gly Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 128 <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 Ala Ala Ser Gly Tyr Pro Tyr Ser Asn Gly 20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Thr Ile Tyr Thr Gly Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Arg Ala Ala Pro Leu Tyr Ser Ser Gly Ser Pro Leu Thr Arg 100 105 110 Ala Arg Tyr Asn Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Thr Tyr Cys Thr Tyr 20 25 30 Asp Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val 35 40 45 Ser Ala Ile Asp Ser Asp Gly Thr Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Gly Thr Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys 85 90 95 Thr Val Cys Val Val Gly Ser Arg Trp Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 6 <211> 117 <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 Thr Leu Ser Cys Ala Ala Ser Glu Tyr Ala Tyr Ser Thr Cys 20 25 30 Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ser Ala Phe Ile Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser 85 90 95 Ala Asn Cys Tyr Arg Arg Leu Arg Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 124 <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 Thr Ala Ser Gly Leu Thr Phe Asp Asp Ser 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Ala Val 35 40 45 Ser Cys Ile Ser Ser Ser Gly Ala Asn Ala Phe 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 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Lys Arg Gly His Ala Cys Ala Gly Tyr Tyr Pro Ile Pro Tyr Asp Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Ala Ile Ala Ser Gly Tyr Ile Asp Ser Arg Trp Cys Met Ala 1 5 10 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Ala Ile Trp Pro Gly Gly Gly Leu Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Gly Ser Pro Arg Met Cys Pro Ser Leu Glu Phe Gly Phe Asp Tyr 1 5 10 15 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Phe Thr Ser Asn Ser Cys Gly Met Asp 1 5 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Ser Ile Ser Thr Asp Gly Thr Thr Gly Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Lys Asp Gly Thr Ile Ala Thr Met Glu Leu Cys Asp Phe Gly Tyr 1 5 10 15 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Tyr Pro Tyr Ser Asn Gly Tyr Met Gly 1 5 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Thr Ile Tyr Thr Gly Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 16 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Arg Ala Ala Pro Leu Tyr Ser Ser Gly Ser Pro Leu Thr Arg Ala Arg 1 5 10 15 Tyr Asn Val <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Ser Thr Tyr Cys Thr Tyr Asp Met His 1 5 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Ala Ile Asp Ser Asp Gly Thr Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Val Cys Val Val Gly Ser Arg Trp Ser Asp Tyr 1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Tyr Ala Tyr Ser Thr Cys Asn Met Gly 1 5 <210> 21 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Ala Phe Ile Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Asn Cys Tyr Arg Arg Leu Arg Asn Tyr 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Leu Thr Phe Asp Asp Ser Val Met Gly 1 5 <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Cys Ile Ser Ser Ser Gly Ala Asn Ala Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 25 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Gly His Ala Cys Ala Gly Tyr Tyr Pro Ile Pro Tyr Asp Asp Tyr 1 5 10 15 <210> 26 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 26 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 Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 27 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 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 Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Val Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asp Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 28 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 28 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 Leu Ser Thr Tyr 20 25 30 Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Thr Val Ser Arg Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Leu Ala Ser Val Ser Asn Leu Gly Trp Pro Pro Val Arg Ala Pro Ser 100 105 110 Pro Thr Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 29 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Ala Gly Thr Tyr Tyr Thr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg His Gly Tyr 100 105 110 Asn Val Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 30 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 30 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 Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Val Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Ile Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asp Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 31 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 Leu Ser Thr Tyr 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ala Val Ser Arg Gly Gly Phe Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Met Ser Ser Val Ser Phe Tyr Gly Trp Pro Pro Asp Arg Val Pro Ser 100 105 110 Pro Thr Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 32 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 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 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Ala Gly Thr Tyr Tyr Thr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Gly Tyr 100 105 110 Asn Val Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Phe Thr Tyr Arg Pro Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Thr Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Thr Thr Val Ile Thr Ser Val Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 34 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 34 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 Gly Ile Ser Gly Phe Thr Tyr Arg Pro Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Ser Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 35 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 35 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 Thr Ala Ser Gly Phe Thr Phe Arg 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 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Arg Leu His Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 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 Arg 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 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Cys Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Gly Tyr 100 105 110 Asn Cys Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 38 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Tyr Ser Gly Ser Ser Thr Tyr Tyr Val Asp Ser Val 50 55 60 Leu Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Gln Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Val Lys Glu Arg Arg Ser Asn Gly His Pro Ile Val Phe Gly 100 105 110 Asp Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 39 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 39 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 Ile Ser Gly Phe Thr Tyr Lys Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 40 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 40 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 Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Val Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asp Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Tyr Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 41 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg 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 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 42 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 Arg 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 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Ala Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 43 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 Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Ala 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Asp Tyr 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 44 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 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 Gly Ile Ser Gly Phe Thr Tyr Arg Pro Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Ser Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Ala Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 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 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Val Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 46 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Gln Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Pro Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 47 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 47 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 Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Gly Gly Gly Ala Thr Tyr 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 Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Asn Leu Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 48 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 48 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 Thr Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 His Met Lys Trp Glu Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Thr Pro Gly Gly Ala Ser Thr Gly 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 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Leu Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 49 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Phe Thr Tyr Arg Pro Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Thr Thr Gly Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Thr Thr Val Ile Thr Ser Val Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 50 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 Ile Ser Gly Phe Thr Tyr Lys Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Thr Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 51 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 Ile Ser Gly Phe Val Tyr Lys Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 52 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 52 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Val Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 53 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser 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 Thr Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 54 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 54 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Ala Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Cys Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Cys Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 55 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 56 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 56 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 Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Val Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asp Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 57 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Val Val Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 58 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 58 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met 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 His Val Ala Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Trp 100 105 110 His Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 59 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 59 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 Arg Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Ala 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 60 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 60 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Ile Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 61 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 61 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Val Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Met Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asp Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 62 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 62 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Pro Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 63 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 63 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Phe Leu Arg Leu Ser Cys Ala Phe Ser Gly Tyr Thr Gly Cys Met Gly 20 25 30 Trp Phe Arg Gln Gly Pro Gly Gln Glu Arg Glu Gly Val Ala Ser Ile 35 40 45 Asn Asp Gly Gly Ser Leu Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Lys Asp Asn Ala Lys Lys Thr Leu Asp Leu Gln Met Asn 65 70 75 80 Thr Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ser Leu 85 90 95 Ser Tyr Cys Leu Asn Pro Thr Leu Arg Val Asp Gly Tyr Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 64 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 64 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 Ile Ser Gly Leu Thr Tyr Lys Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Tyr Leu Tyr 100 105 110 Gln Trp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 65 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 65 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Ser Val Phe Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Leu Ser Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Gly Tyr Asn 100 105 110 Val Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 66 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 66 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 Gly Ile Ser Gly Phe Thr Tyr Arg Pro Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asp Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 67 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 67 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Gly Tyr 100 105 110 Asn Val Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 68 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 68 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Leu Gly Gly Asp Thr Thr Tyr Tyr Thr 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 Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Lys His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Gly Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 69 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 69 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 His Leu Lys Trp Glu Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Thr Asn Gly Gly Ala Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Leu Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 70 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 70 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Val Ile Ser Gly Phe Thr Tyr Arg Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Ile Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 71 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 71 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 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr 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 Thr Glu Asp Thr Ala Met Tyr Cys Cys 85 90 95 Ala Lys His Ile Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr 100 105 110 Asn Cys Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 72 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 72 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 Ile Ser Gly Phe Thr Tyr Lys Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Asn Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Thr Val Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 73 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 73 Gln Val Gln Leu Gln Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Gly Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Asn Asp Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gln Asn Leu Asp Tyr Arg Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 74 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 74 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 Ile Ser Gly Phe Thr Tyr Lys Gln Thr 20 25 30 Phe Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Glu Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Gly Leu Lys Leu Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Val Ile Thr Ser Val Thr Ile Asn Arg Gly Leu Tyr 100 105 110 Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 75 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 Phe Thr Tyr Arg Gln Thr Phe Met Gly 1 5 <210> 76 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 76 Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 77 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 77 Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr Gln Tyr 1 5 10 15 <210> 78 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Phe Thr Tyr Arg Gln Thr Phe Met Gly 1 5 <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 80 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr Gln Tyr 1 5 10 15 <210> 81 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 81 Phe Thr Leu Ser Thr Tyr Trp Ile Tyr 1 5 <210> 82 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 82 Thr Val Ser Arg Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Asn 1 5 10 15 Gly <210> 83 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Ser Val Ser Asn Leu Gly Trp Pro Pro Val Arg Ala Pro Ser Pro 1 5 10 15 <210> 84 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 <210> 85 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Ala Ile Asn Ser Gly Gly Ala Gly Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 86 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg His Gly Tyr Asn Val 1 5 10 15 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 Phe Thr Tyr Arg Gln Thr Phe Met Gly 1 5 <210> 88 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Ala Ile Ser Thr Gly Gly Gly Ser Thr Ile Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 89 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Ser Thr Val Ile Thr Ser Glu Ser Ile Asn Arg Asn Leu Tyr Gln Tyr 1 5 10 15 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Phe Thr Leu Ser Thr Tyr Trp Met Tyr 1 5 <210> 91 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Ala Val Ser Arg Gly Gly Phe Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 92 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Ser Val Ser Phe Tyr Gly Trp Pro Pro Asp Arg Val Pro Ser Pro 1 5 10 15 <210> 93 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 <210> 94 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 94 Ala Ile Asn Ser Gly Gly Ala Gly Thr Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly <210> 95 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 95 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Gly Tyr Asn Val 1 5 10 15 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 96 Phe Thr Tyr Arg Pro Thr Phe Met Gly 1 5 <210> 97 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 97 Ala Ile Thr Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 98 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 98 Thr Thr Val Ile Thr Ser Val Ser Ile Asn Arg Asn Leu Tyr Gln Tyr 1 5 10 15 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 99 Phe Thr Tyr Arg Pro Thr Phe Met Gly 1 5 <210> 100 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 100 Ala Ile Ser Thr Gly Gly Gly Ser Ser Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 101 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 101 Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr Gln Tyr 1 5 10 15 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 102 Phe Thr Phe Arg Asn Tyr Ala Met Ser 1 5 <210> 103 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 104 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 104 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr Asn Tyr 1 5 10 15 <210> 105 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 105 Phe Thr Phe Arg Asn Tyr Ala Met Ser 1 5 <210> 106 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 106 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 107 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 107 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr Asn Tyr 1 5 10 15 <210> 108 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 <210> 109 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 109 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 110 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 110 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Gly Tyr Asn Cys 1 5 10 15 <210> 111 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 111 Phe Thr Phe Ser Thr Tyr Asp Met Ser 1 5 <210> 112 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 112 Thr Ile Asn Tyr Ser Gly Ser Ser Thr Tyr Tyr Val Asp Ser Val Leu 1 5 10 15 Gly <210> 113 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 113 Val Lys Glu Arg Arg Ser Asn Gly His Pro Ile Val Phe Gly Asp 1 5 10 15 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 114 Phe Thr Tyr Lys Gln Thr Phe Met Gly 1 5 <210> 115 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 115 Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 116 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 116 Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Gly Leu Tyr Gln 1 5 10 15 <210> 117 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 117 Phe Thr Tyr Arg Gln Thr Phe Met Gly 1 5 <210> 118 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 118 Ala Ile Ser Thr Gly Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 119 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 119 Ser Thr Val Ile Thr Ser Glu Ser Ile Tyr Arg Asn Leu Tyr Gln Tyr 1 5 10 15 <210> 120 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 120 Phe Thr Phe Arg Asn Tyr Ala Met Ser 1 5 <210> 121 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 121 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 122 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 122 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr Asn Tyr 1 5 10 15 <210> 123 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 123 Phe Thr Phe Arg Asn Tyr Ala Met Ser 1 5 <210> 124 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 124 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 125 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 125 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr Ala Tyr 1 5 10 15 <210> 126 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 126 Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 <210> 127 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 127 Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Ala <210> 128 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 128 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Asp Tyr Asn Tyr 1 5 10 15 <210> 129 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 129 Phe Thr Tyr Arg Pro Thr Phe Met Gly 1 5 <210> 130 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 130 Ala Ile Ser Thr Gly Gly Gly Ser Ser Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 131 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 131 Ser Thr Val Ile Thr Ser Val Ser Ile Asn Arg Ala Leu Tyr Gln Tyr 1 5 10 15 <210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 132 Phe Thr Phe Ser Ser Tyr Ala Val Ser 1 5 <210> 133 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 133 Thr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 134 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 134 His Val Thr Gly Asp Tyr Asp Pro Ser Leu Arg Tyr Glu Tyr Asn Val 1 5 10 15 <210> 135 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 135 Phe Thr Tyr Arg Gln Thr Phe 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 Thr Gly Gly Gly Ser Thr Val 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 Ser Thr Val Ile Thr Ser Pro Ser Ile Asn Arg Asn Leu Tyr Gln Tyr 1 5 10 15 <210> 138 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 138 Phe Thr Phe Ser Asn Tyr Ala Met Lys 1 5 <210> 139 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 139 Ser Ile Ser Gly Gly Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 140 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 140 Gln Asn Leu Asp Tyr 1 5 <210> 141 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 141 Phe Thr Phe Asn Ser Ala His Met Lys 1 5 <210> 142 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 142 Phe Ile Thr Pro Gly Gly Ala Ser Thr Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 143 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 143 Gly Gly Leu 1 <210> 144 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 144 Phe Thr Tyr Arg Pro Thr Phe Met Gly 1 5 <210> 145 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 145 Ala Ile Thr Thr Gly Gly Gly Ser Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 146 <211> 16 <212> PRT ...

Claims

1. A gp130-binding molecule that specifically binds to the extracellular domain of gp130.

2. A gp130-binding molecule according to claim 1, comprising a single-domain antibody (sdAb).

3. sdAb is shown in the following table: The gp130 binding molecule according to claim 2, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3 as shown in the row.

4. The gp130-binding molecule according to claim 2 or 3, 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 respect to any one polypeptide sequence of SEQ ID NO: 2, 3, 4, 5, 6, and 7.

5. sdAb is shown in the following table: The gp130 binding molecule according to claim 2, comprising complementarity-determining regions 1 (CDR1), CDR2, and CDR3 as shown in the row.

6. The gp130 binding molecule according to claim 2, 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: 26 to 74.

7. The gp130-binding molecule according to any one of claims 3 or 5, comprising a humanized or otherwise CDR grafted onto a heterogeneous framework.

8. A gp130-binding molecule according to any one of claims 1 to 7, further comprising a labeling agent, an imaging agent, and / or a therapeutic agent.

9. A method for treating or preventing a disease, disorder, or condition in a mammalian subject by administering a therapeutically effective amount of the gp130 binding molecule or a pharmaceutically acceptable formulation thereof according to any one of claims 1 to 8 to the mammalian subject.

10. The method according to claim 9, wherein the disease is a neoplastic disease.

11. A gp130-binding molecule according to any one of claims 1 to 8, for use in the isolation, depletion, or enrichment of gp130+ cells from a biological sample.

12. A nucleic acid sequence encoding the gp130 binding molecule according to any one of claims 1 to 8.

13. A recombinant viral vector or nonviral vector comprising the nucleic acid described in claim 12.

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

15. A pharmaceutical formulation comprising a viral vector or non-viral vector as described in claim 13.

16. A kit comprising a gp130-binding molecule according to any one of claims 1 to 8.

Images