IL27Rα-binding molecules and methods of use

Abstract

The present disclosure relates to biologically active molecules, including single domain antibodies (sdAbs) that specifically bind to the extracellular domain of human IL27Rα, compositions comprising such antibodies, and methods of use thereof.

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 incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present disclosure relates to biologically active molecules, including single domain antibodies that specifically bind to the extracellular domain of IL27 receptor alpha (IL27Rα), compositions comprising such single domain antibodies, and methods of use thereof. [Background technology]

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

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

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

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

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

[0007] Monoclonal antibodies are the most widely used reagents for protein detection and quantification, but their size can potentially limit their use in assays using several reagents competing for nearby epitope recognition. A unique immunoglobulin class containing a heavy chain domain and lacking a light chain domain (commonly referred to as "heavy chain" antibodies (HCAbs)) is present in camelids, including dromedaries, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicuñas, and guanacos, as well as cartilaginous fish such as sharks. Isolated variable domain regions of HCAbs are known as VHHs (an abbreviation for "variable-heavy-heavy," reflecting their structure) or Nanobodies® (Ablynx). Single-domain VHH antibodies have the advantage of being small (approximately 12–14 kD), approximately one-tenth the molecular weight of conventional mammalian IgG class antibodies, which facilitates binding of these VHH molecules to target antigenic determinants that may be inaccessible to conventional monoclonal IgG formats (Ingram et al., 2018). Furthermore, VHH single-domain antibodies often feature high thermal stability, facilitating drug delivery to regions where cold chain maintenance is difficult or impossible. These properties, especially when combined with simple phage display recovery methods that do not require heavy / light chain pairing (as is the case with IgG antibodies) and simple production (e.g., in bacterial expression systems), make VHH single-domain antibodies useful for a variety of applications, including the development of imaging and therapeutic agents. Summary of the Invention

[0008] The present disclosure provides polypeptides that specifically bind to IL27Ra.

[0009] The present disclosure provides polypeptides that specifically bind to the extracellular domain of IL27Ra.

[0010] The present disclosure provides IL27Ra-binding molecules that specifically bind to the extracellular domain of human IL27Ra (hIL27Ra).

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

[0012] In some embodiments, the IL27Ra binding molecule is an sdAb, and the sdAb comprises a set of CDRs corresponding to CDR1, CDR2, and CDR3 as shown in a row in Table 1 below.

[0013] In some embodiments, an IL27Ra-binding molecule comprises CDR1, CDR2, and CDR3 as set forth in a row of Table 1 below, and CDR1, CDR2, and CDR3 may each independently comprise at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence set forth in a row of Table 1 below, and may have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

[0014] In some embodiments, the IL27Ra-binding molecule consists of, optionally consists essentially of, or optionally comprises a single domain antibody (sdAb) having at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 98%, alternatively at least 99% identity (or identical except for 1, 2, 3, or 4 amino acids that are optionally conservative amino acid substitutions), or alternatively 100% identity to the polypeptide sequence of any one of SEQ ID NOs:2-25, as set forth in Table 1 below.

[0015] (Table 1) TIFF0007652883000001.tif60160TIFF0007652883000002.tif235160TIFF0007652883000003.tif235160TIFF0007652883000004.tif204160

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

[0017] Additionally, the present disclosure provides chemical or recombinant process methods for preparing the IL27Ra binding molecules of the present disclosure.

[0018] Additionally, the present disclosure provides nucleic acids encoding IL27Ra-binding molecules. Table 2 below shows examples of DNA sequences that encode IL27Ra-binding molecules as described herein.

[0019] (Table 2) DNA sequences encoding the VHHs in Table 1 TIFF0007652883000005.tif202158TIFF0007652883000006.tif202158TIFF00076528830 00007.tif202158TIFF0007652883000008.tif202158TIFF0007652883000009.tif163158

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

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

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

[0023] In some embodiments, an IL27Ra-binding molecule comprises CDR1, CDR2, and CDR3 as set forth in a row of Table 3 below, and CDR1, CDR2, and CDR3 may each independently comprise at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity compared to the sequence set forth in a row of Table 3 below, and may have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

[0024] In some embodiments, the IL27Ra-binding molecule consists of, optionally consists essentially of, or optionally comprises a single domain antibody (sdAb) having at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 98%, alternatively at least 99% identity (or identical except for 1, 2, 3, or 4 amino acids that are optionally conservative substitutions), or 100% identity to the polypeptide sequence of any one of SEQ ID NOs: 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, and 174, as set forth in Table 3 below.

[0025] Table 3: mIL2Rg VHH and CDR amino acid (AA) sequences TIFF0007652883000010.tif135167TIFF0007652883000011.tif216167TIFF0007652883000012.tif216167TIFF0007652883000013.tif54167

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

[0027] Additionally, the present disclosure provides chemical or recombinant process methods for preparing the IL27Rα binding molecules of the present disclosure.

[0028] Additionally, the present disclosure provides nucleic acids encoding IL27Ra-binding molecules. Table 4 below shows examples of DNA sequences encoding hIL27Ra-binding molecules, as described in Table 3 above.

[0029] Table 4: DNA sequences encoding the VHHs in Table 3 TIFF0007652883000014.tif91164TIFF0007652883000015.tif212164TIFF0007652883000016.tif212164TIFF0007652883000017.tif91164

[0030] Additionally, the present disclosure provides recombinant viral and non-viral vectors comprising nucleic acids encoding an IL27Rα-binding molecule of the present disclosure or a CDR of an IL27Rα-binding molecule of the present disclosure.

[0031] Additionally, the present disclosure provides host cells comprising recombinant viral and non-viral vectors that comprise nucleic acids encoding an IL27Rα-binding molecule of the present disclosure or a CDR of an IL27Rα-binding molecule of the present disclosure.

[0032] Additionally, the present disclosure provides host cells comprising recombinant viral and non-viral vectors that comprise nucleic acids encoding an IL27Rα-binding molecule of the present disclosure or a CDR of an IL27Rα-binding molecule of the present disclosure.

[0033] Additionally, the present disclosure provides kits comprising the IL27Rα-binding molecules of the present disclosure.

[0034] In another aspect, the present disclosure provides a construct for identifying IL27Rα-expressing cells, wherein the IL27Rα-binding molecule is conjugated to one or more imaging agents, optionally via a chemical or polypeptide linker. Furthermore, the present disclosure provides a method of use of the aforementioned in identifying IL27Rα-expressing cells in a subject, comprising administering to a subject in need of treatment an effective amount of an IL27Rα-binding molecule conjugated to an imaging agent, and evaluating the subject for the presence of the imaging agent conjugated to the IL27Rα-binding molecule.

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

[0036] The present disclosure provides IL27Rα-binding molecules comprising a polypeptide sequence that specifically binds to the extracellular domain of IL27Rα, and methods for their use in isolating, depleting, or enriching IL27Rα-expressing cells in a biological sample. [The present invention 1001] An IL27Rα-binding molecule that specifically binds to the extracellular domain of IL2Rb. [The present invention 1002] 1001. The IL27Rα-binding molecule of the present invention, wherein the IL2Rb-binding molecule comprises a single domain antibody (sdAb). [The present invention 1003] The sdAb is listed in the following table: TIFF0007652883000018.tif177167TIFF0007652883000019.tif151167 1002. An IL27Rα-binding molecule of the present invention, comprising complementarity determining region 1 (CDR1), CDR2, and CDR3 as shown in the row. [The present invention 1004] The IL27Rα-binding molecule of the present invention 1002 or 1003, wherein the sdAb has at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% identity to the polypeptide sequence of any one of SEQ ID NOs: 2-25. [The present invention 1005] The sdAb is listed in the following table: TIFF0007652883000020.tif84170 1002. An IL27Rα-binding molecule of the present invention, comprising complementarity determining region 1 (CDR1), CDR2, and CDR3 as shown in the row. [The present invention 1006] 1002. The IL27Rα binding molecule of the present invention, wherein the sdAb has at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% identity to the polypeptide sequence of any one of SEQ ID NOs: 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, and 174. [The present invention 1007] IL27Rα binding molecule of either 1003 or 1005, wherein the sdAb is humanized or otherwise comprises CDRs grafted onto a heterologous framework. [The present invention 1008] The IL27Rα-binding molecule of any of claims 1001 to 1007, further comprising a labeling agent, an imaging agent, and / or a therapeutic agent. [The present invention 1009] 1009. The IL27Rα-binding molecule of any of claims 1001 to 1008 for use in isolating, depleting, or enriching IL27Rα+ cells from a biological sample. [The present invention 1011] A nucleic acid sequence encoding any one of the IL27Rα-binding molecules of the present invention 1001 to 1008. [The present invention 1012] A recombinant viral or non-viral vector comprising a nucleic acid of the present invention. [The present invention 1013] A host cell comprising a nucleic acid of the present invention. [The present invention 1014] A kit comprising the IL2Rb-binding molecule of any one of 1001 to 1008 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention Introduction In order to more readily understand this disclosure, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be interpreted in light of the knowledge known to those of ordinary skill in the art.

[0038] Before the present methods and compositions are described, it is to be understood that this disclosure is not limited to the particular method or composition described, as such may, of course, vary.

[0039] Where a range of values ​​is stated, it is understood that each intervening value between the upper and lower limits of that range is specifically disclosed to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each narrower range between any stated or intervening value within a stated range and any other stated or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these narrower ranges may independently be included or excluded within the range, and each range that includes one limit, neither limit, or both limits in the narrower range is also encompassed within the invention, subject to any limits explicitly excluded in the stated range. When a stated range includes one or both limits, ranges excluding either or both of the included limits are also encompassed within the invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of this invention, some possible and preferred methods and materials will now be described.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials described in the cited publications.

[0041] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, e.g., polypeptides, etc.

[0042] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0043] It will be understood that throughout this disclosure, amino acids will be referred to according to their single-letter or three-letter codes. For the convenience of the reader, the single-letter and three-letter amino acid codes are provided below in Table 5.

[0044] Table 5: Amino acid abbreviations TIFF0007652883000021.tif110128

[0045] Standard methods in molecular biology are described in the scientific literature (see, e.g., 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, which describe 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)). The scientific literature describes protein purification methods including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

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

[0047] Activate: As used herein, the term "activate" is used with respect to a receptor or receptor complex to reflect a biological effect directly and / or by participation in a multi-component signaling cascade resulting from the binding of an agonist ligand to the receptor in response to ligand binding.

[0048] Active: 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 of the molecule to another molecule) or physical properties (e.g., alteration 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, intracellular signaling, gene expression, the ability to stimulate cell proliferation, and the ability to modulate immunological activities such as inflammatory responses. "Activity" is typically expressed as the level of biological activity per unit of the test agent, e.g., [catalytic activity] / [mg protein], [immunological activity] / [mg protein], International Units of activity (IU), [STAT5 phosphorylation] / [mg protein], [proliferation] / [mg protein], plaque-forming units (pfu), etc. The term proliferative activity, as used herein, refers to the activity of promoting cell growth and replication, including dysregulated cell division, e.g., dysregulated cell division observed in neoplastic diseases, inflammatory diseases, fibrosis, metaplasia, cellular transformation, metastasis, and angiogenesis.

[0049] Administer / Administer: The terms "administration" and "administering" are used interchangeably herein to refer to the act of contacting a subject, including contacting the subject's cells, tissues, organs, or biological fluids in vitro, in vivo, or ex vivo with an agent (e.g., an IL27Rα-binding molecule or engineered cells expressing an IL27Rα-binding molecule, a chemotherapeutic agent, an antibody, or a pharmaceutical formulation containing one or more of the foregoing). Administration of an agent can be accomplished by any of a variety of art-recognized methods, 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, iontophoretic delivery, intralymphatic injection, intragastric injection, intraprostatic injection, intravesical injection (e.g., bladder), inhalation (e.g., respiratory inhaler, including dry powder inhaler), intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), etc. The term "administration" includes contact of an agent with a cell, tissue, or organ, as well as contact of an agent with a fluid in contact with a cell, tissue, or organ.

[0050] Affinity: As used herein, the term "affinity" refers to the degree of specific binding between a first molecule (e.g., a ligand) and a second molecule (e.g., a receptor), and is expressed as the dissociation rate constant (k off ) and the association rate constant (k on ) and the equilibrium dissociation constant K D It is measured by

[0051] Agonists: As used herein, the term "agonist" refers to a first agent that specifically binds to a second agent (a "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 cell activation, enhances activation, increases the cell's sensitivity to activation by a second agent, or upregulates the expression of one or more genes, proteins, ligands, receptors, biological pathways that may lead to cell proliferation, or pathways that lead to cell cycle arrest or cell death, e.g., cell death by apoptosis. In some embodiments, an agonist is an agent that binds to a receptor, changes the receptor state, and thereby produces 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 referred to as 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 associated with the natural ligand / receptor binding interaction). A "superagonist" is a type of agonist that can produce a maximum response that exceeds that of an endogenous agonist at a target receptor, and thus has more than 100% of the activity of a native ligand. A superagonist is typically a synthetic molecule that, when evaluated at similar concentrations in a comparable assay, exhibits more than 110%, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, or more than 170% of the response of an evaluable quantitative or qualitative parameter of a natural molecule. It should be noted that the biological effects associated with a full agonist may differ in degree and / or type from those of a partial agonist or superagonist. In contrast to agonists, antagonists can specifically bind to receptors but do not trigger a signal cascade, typically initiated by the receptor, and may alter the agonist action at that receptor. An inverse agonist is an agent that produces a pharmacological response that is opposite in direction to that of an agonist.

[0052] Antagonists: As used herein, the term "antagonist" or "inhibitor" refers to a molecule that opposes the action of an agonist.Antagonists block, reduce, inhibit, or neutralize the activity of agonists, and can also block, inhibit, or reduce the constitutive activity of targets, such as target receptors, even in the absence of a specific agonist.An inhibitor is, for example, a molecule that reduces, blocks, inhibits, or delays the activation of a biological pathway, including a gene, protein, ligand, receptor, immune checkpoint pathway, or cell, or that inactivates, desensitizes, or downregulates a biological pathway, including a gene, protein, ligand, receptor, immune checkpoint pathway, or cell.

[0053] antibody: As used herein, the term "antibody" collectively refers to (a) glycosylated or non-glycosylated immunoglobulins that specifically bind to a target molecule, and (b) immunoglobulin derivatives thereof, including, but not limited to, antibody fragments such as single-domain antibodies. In some embodiments, immunoglobulin derivatives compete with the immunoglobulin of origin for binding to a target molecule. The term antibody is not limited to immunoglobulins derived from any particular species and includes antibodies from murine, human, equine, camelid, 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 monoclonal, bispecific, trispecific, chimeric, humanized, human, CDR-grafted, veneered, or deimmunized (e.g., to remove T-cell epitopes), camelized (in the case of VHH), or engineered antibodies, including molecules comprising the binding domain (e.g., CDR) of an antibody in a non-immunoglobulin scaffold. The term "antibody" should not be construed as limited to any particular synthetic means and includes natural antibodies that can be isolated from natural sources, as well as engineered 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 that result in the expression of antibodies, and antibodies isolated from combinatorial antibody libraries, including phage display libraries. In one embodiment, the "antibody" is a mammalian immunoglobulin of the IgG1, IgG2, IgG3, or IgG4 class. In some embodiments, the antibody is a "full-length antibody" comprising 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 can specifically bind to an antigen and compete for binding with the parent antibody from which it was derived. The term "single-domain antibody" includes scFv and VHH molecules.As used herein, the term "VHH" refers to single domain antibodies derived from camelid antibodies, typically obtained from immunization of camelids (including camels, llamas, and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448). VHHs are also referred to as heavy chain antibodies or Nanobodies®. Single domain antibodies may also be derived from non-mammalian sources, such as VHHs obtained from IgNAR antibody immunization of cartilaginous fish, including, but not limited to, sharks.

[0054] Biological samples: As used herein, the term "biological sample" or "sample" refers to a sample obtained (or derived) from a subject. By way of example, a biological sample includes material selected from the group consisting of bodily fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (e.g., vitreous humor, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including immunoglobulin-enriched or cell type-specific enriched fractions derived from one or more of such tissues.

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

[0056] CDR: As used herein, the term "CDR" or "complementarity-determining region" is intended to refer to the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain immunoglobulin polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, et al., U.S. Department of Health and Human Services publication entitled "Sequences of proteins of immunological interest" (1991) (also referred to herein as "Kabat 1991" or "Kabat"); Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (also referred to herein as "Chothia"); and MacCallum, et al. (1996) J. Mol. Biol. 262:732-745, and the definition includes overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. In the context of this disclosure, unless otherwise specified, CDR positions are numbered according to Kabat, or a hybrid of the Kabat and Chothia numbering conventions.

[0057] Equivalent: As used herein, the term "equivalent" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements would be considered "equivalent" if a first measurement of an evaluable quantitative parameter and a second measurement of the evaluable parameter do not deviate beyond a range that one skilled in the art would recognize as not resulting in a statistically significant difference in effect between the two results in this situation. In some cases, measurements may be considered "equivalent" if one measurement deviates from another measurement by less than 35%, alternatively less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or alternatively less than 1%. In certain embodiments, a measurement is equivalent to a standard if it deviates from the standard by less than 15%, alternatively less than 10%, or alternatively less than 5%.

[0058] Conservative amino acid substitutions: As used herein, the term "conservative amino acid substitution" refers to an amino acid exchange in which a particular amino acid is changed for another amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, amino acids in each of the following groups can be considered conservative amino acids: (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.

[0059] Derived from: As used herein, the term "derived from," in the context of an amino acid sequence, is intended to indicate that a polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid sequence, and is not intended to be limiting with respect to the source or manner in which the protein or nucleic acid is made. By way of example, the term "derived from" includes homologs or variants of the reference amino acid or DNA sequence.

[0060] Effective concentration (EC): As used herein, the term "effective concentration" or its abbreviation "EC" is used interchangeably to refer to an agent concentration sufficient to alter a particular parameter in a test system. The abbreviation "E" refers to the magnitude of a particular biological effect observed in a test system when the test system is exposed to a test agent. The abbreviation "EC" is used when the magnitude of the response is expressed as a factor of the concentration ("C") of the test agent. In the context of biological systems, the term Emax refers to the maximum magnitude of a particular biological effect observed in response to a saturating concentration of an activating test agent. When the abbreviation EC is presented with a subscript (e.g., EC 40 , E.C. 50 etc.), the subscript refers to the percent Emax of the biological response observed at that concentration. For example, a concentration of a test agent sufficient to induce a measurable biological parameter in a test system that is 30% of the maximal level of such measurable biological parameter in response to such test agent is referred to as the "EC 30 Similarly, it is called "EC 100 The term EC2 (commonly used in the field of pharmacokinetics) is used to denote the effective concentration of an agent that produces a maximal (100%) response of a measurable parameter in response to such an agent. 50The term "saturation concentration" refers to the concentration of an agent sufficient to produce a half-maximal (approximately 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 a drug such that all available receptors are occupied by the drug, and is referred to as the EC 50 is the drug concentration that produces the half-maximal effect.

[0061] Concentrated: As used herein, the term "enriched" refers to a sample that is non-naturally engineered such that the species of interest (e.g., molecule or cell) is present at a concentration that is higher (e.g., at least 3-fold, alternatively at least 5-fold, alternatively at least 10-fold, alternatively at least 50-fold, alternatively at least 100-fold, or alternatively at least 1000-fold) than the concentration of the species in the starting sample, e.g., biological sample (e.g., the sample in which the molecule naturally occurs or the sample in which it occurs after administration), or (b) a sample that has been non-naturally engineered such that the species is present at a concentration that is higher than the environment in which the molecule was made (e.g., recombinantly modified bacterial or mammalian cells).

[0062] Extracellular domain: As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is on the outside of the plasma membrane of a cell. A cell surface protein may be a transmembrane protein, a cell surface protein, or a membrane-bound protein.

[0063] Identity: The term "identity," as used herein with respect to polypeptide or DNA sequences, refers to subunit sequence identity between two molecules. If a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide), the molecules are identical at that position. The similarity between two amino acid sequences or two nucleotide sequences is a linear function of the number of identical positions. Generally, the 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 analyses is publicly available through the website of the National Center for Biotechnology Information (NCBI). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or fit a positive threshold score "T." T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.Extension of word hits in each direction stops when (a) the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score becomes 0 or less due to the accumulation of one or more negative-scoring residue alignments; or (b) the end of either sequence is reached. 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 as defaults a word size ("W") of 28, an expectation ("E") of 10, M=1, N=-2, and comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size ("W") of 3, an expectation (E") of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS (USA) 89:10915-10919).

[0064] In sufficient quantity to bring about a response: As used herein, the phrase "in an amount sufficient to elicit a response" refers to an amount of a test agent sufficient to produce a detectable change between the level of an indicator measured before (e.g., a baseline level) and the level of an indicator measured after application of the test agent to the test system. 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 fluorescent assay. In some embodiments, the test system is an in vivo system involving measurement of a change in the level of a parameter of a cell, tissue, or organism that reflects the biological function before and after application of a test agent to the cell, tissue, or organism. In some embodiments, the indicator reflects the biological function or developmental state of the cell assessed in the assay in response to administration of an amount of the test agent. In some embodiments, the test system involves measurement of a change in the level of an indicator of a cell, tissue, or organism that reflects the biological state before and after application of one or more test agents to the cell, tissue, or organism. The term "in an amount sufficient to elicit a response" may be a therapeutically effective amount, but may be greater or less than a therapeutically effective amount.

[0065] Inhibitors: The term "inhibitor" as used herein refers to a molecule that, for example, decreases, blocks, prevents, delays the activation of a gene, protein, ligand, receptor, or cell, or inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity of a cell or organism.

[0066] Intracellular domain: As used herein, the term "intracellular domain" or its abbreviation "ICD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is within the plasma membrane of the cell. The ICD may include the entire cytoplasmic portion of a transmembrane or membrane-bound protein, or may include an intracellular protein.

[0067] Isolated: The term "isolated" used herein refers to a polypeptide of interest that is in an environment different from the environment in which it may naturally exist if it exists in nature. "Isolated" is intended to include the polypeptide in a sample that is highly enriched for the polypeptide of interest and / or the polypeptide of interest is partially or substantially purified.If the polypeptide is not natural, "isolated" refers to the polypeptide being separated from the synthetic environment, for example, being isolated from a recombinant cell culture that contains cells that are engineered to express the polypeptide, or being isolated by a solution resulting from solid-phase synthesis.

[0068] Kabat numbering: As used herein, the term "Kabat numbering" is art-recognized and refers to a system for numbering amino acid residues that are more variable (e.g., hypervariable) than other amino acid residues in the heavy and light chain regions of immunoglobulins (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 art-recognized and refers to a system for numbering amino acid residues based on their location in structural loop regions (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For purposes of this disclosure, unless specifically specified otherwise, the locations of CDR2 and CDR3 in the variable region of an antibody are according to Kabat numbering, or simply "Kabat." The location of CDR1 in the variable region of an antibody is according to a hybrid of the Kabat and Chothia numbering schemes.

[0069] Ligand: The term "ligand" as used herein refers to a molecule that specifically binds to a receptor and induces a change in the receptor that alters the activity of the receptor or the response of a cell that expresses that receptor. In one aspect, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of the receptor. As used herein, the term "ligand" encompasses natural and synthetic ligands. "Ligand" also encompasses small molecules, cytokines, and peptide mimetics of antibodies. A complex of a ligand and receptor is called a "ligand-receptor complex." A ligand may comprise one domain of a polyprotein or fusion protein (e.g., either domain of an antibody / ligand fusion protein).

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

[0071] Nucleic acid: The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0072] Functionally linked: The term "operably linked" is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, arranged in a construct such that the function of each of the component molecules is maintained, but the operably linked structure can positively or negatively modulate the activity of the individual components of the construct. For example, operably linking a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct in which the biological activity of that protein is reduced relative to the wild-type molecule. However, the two are still considered operably linked. When the term "operably linked" is applied to the relationship between multiple nucleic acid sequences encoding different functions, when the multiple nucleic acid sequences are combined into a single nucleic acid molecule, e.g., when the nucleic acid molecule is introduced into a cell using recombinant techniques, it provides a nucleic acid capable of transcribing and / or translating the particular nucleic acid sequence in the cell. For example, a nucleic acid sequence encoding a signal sequence facilitating secretion of the polypeptide may be considered operably linked to DNA encoding the polypeptide if it expresses a preprotein. A promoter or enhancer may be considered operably linked to a coding sequence if it affects the transcription of the sequence. Alternatively, a sequence is considered operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, in the context of nucleic acid molecules, the term "operably linked" means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader or linked subdomain of the molecule, contiguous and in reading phase. However, certain genetic elements, such as enhancers, can function apart from the sequence they effect and need not be contiguous with that sequence but can still be considered operably linked.

[0073] Parent Polypeptide: As used herein, the terms "parent polypeptide" or "parent protein" are used interchangeably to designate the source of a second polypeptide (e.g., a derivative, mutein, or variant) that is modified relative to a first "parent" polypeptide. In some cases, the parent polypeptide is a wild-type or naturally occurring protein. In some cases, the parent polypeptide may be a modified version of a naturally occurring protein that has been further modified. The term "parent polypeptide" can refer to the polypeptide itself or to a composition comprising the parent polypeptide (e.g., a glycosylated or PEGylated version and / or a fusion protein comprising the parent polypeptide).

[0074] Partial agonists: The term "partial agonist" as used herein refers to a molecule that specifically binds to and activates a particular receptor, but only partially activates the receptor compared to a full agonist. A partial agonist may exhibit both agonist and antagonist 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, thereby acting as a competitive antagonist by net reducing receptor activation compared to the contact of the receptor with 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 the receptor to produce a desired submaximal response in the subject. Alternatively, when an excess amount of endogenous ligand is present, a partial agonist can reduce overstimulation of the receptor. The maximal response (E) produced by a partial agonist can be reduced. max) is referred to as its intrinsic activity and is sometimes expressed on a percentage scale where a full agonist would produce a 100% response. A partial agonist may have more than 10% but less than 100%, 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% of the activity of the reference polypeptide when evaluated at similar concentrations in a particular assay system.

[0075] Polypeptides: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include amino acids specified by the genetic code and amino acids not specified by the genetic code, amino acids that have been chemically or biochemically modified or derivatized, and polypeptides with modified polypeptide backbones. The term polypeptide includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins with an amino acid sequence that facilitates purification, such as a chelating peptide; fusion proteins with an immunologically tagged protein; fusion proteins containing a peptide with an immunologically active polypeptide fragment (e.g., an antigenic diphtheria or tetanus toxin or toxoid fragment); and the like.

[0076] Receptor: As used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds to a ligand, wherein binding of the ligand alters at least one biological property of the polypeptide. In some embodiments, a receptor is a cell membrane-associated protein comprising an extracellular domain (ECD) and a membrane-associated domain that serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide in which the intracellular domain (ICD) and the extracellular domain (ECD) are connected by a transmembrane domain, commonly referred to as a transmembrane domain (TM). Binding of a cognate ligand to a receptor results in a conformational change in the receptor, resulting in a measurable biological effect. In some cases, when a receptor is a transmembrane polypeptide comprising an ECD, a TM, and an ICD, binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to binding of the ligand to the ECD. In some embodiments, a receptor is a component of a multicomponent complex that facilitates intracellular signal transduction. For example, a ligand may bind to a cell surface receptor that, alone, is not involved in any intracellular signaling, but upon ligand binding, promotes the formation of a heteromultimeric (including heterodimers, heterotrimers, etc.) or homomultimeric (including homodimers, homotrimers, homotetramers, etc.) complex, resulting in a measurable biological effect within the cell, e.g., activation of an intracellular signaling cascade (e.g., the Jak / STAT pathway). In some embodiments, the receptor is a single transmembrane polypeptide comprising ECD, TM, and ICD domains, wherein the ECD, TM, and ICD domains are derived from the same or different naturally occurring receptor variants or synthetic functional equivalents thereof.

[0077] Recombination: As used herein, the term "recombinant" is used as an adjective to refer to the way in which a polypeptide, nucleic acid, or cell has been modified 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 way in which the protein was produced (e.g., recombinantly produced human growth hormone is commonly abbreviated as "rhGH"). Similarly, a cell is referred to as a "recombinant cell" if it has been modified by the incorporation (e.g., transfection, transduction, infection) of exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.) using recombinant DNA technology. Techniques and protocols for recombinant DNA technology, such as those found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals, are well known in the art.

[0078] response: For example, the term "response" of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in measurable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, differentiation level or state), which correlate with activation, stimulation, or treatment by, or contact with, an internal mechanism, such as an exogenous agent or genetic programming. In certain circumstances, the terms "activation," "stimulation," and the like refer to cell activation as regulated by internal mechanisms and as regulated by external or environmental factors, while the terms "inhibition," "downregulation," and the like refer to the opposite effect. A "response" may be evaluated in vitro, for example, by using assay systems, surface plasmon resonance, enzyme activity, mass spectroscopy, amino acid or protein sequencing techniques. "Response" may be assessed quantitatively in vivo by assessing objective physiological parameters, e.g., body temperature, body weight, tumor burden, blood pressure, or the results of X-ray or other imaging techniques, or qualitatively by changes in reported subjective feelings of happiness, depression, agitation, or pain. In some embodiments, the level of proliferation of CD3-activated primary human T cells may be assessed in a bioluminescence assay that generates a luminescent signal proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in culture, as described in Crouch, et al. (1993) J. Immunol. Methods 160: 81-8, or using commercially available assays, such as the CellTiter-Glo® 2.0 Cell Viability Assay or the CellTiter-Glo® 3D Cell Viability kit, available from Promega Corporation, Madison, WI 53711, catalog numbers G9241 and G9681, substantially following the instructions provided by the manufacturer.In some embodiments, the level of T cell activation in response to administration of a test agent may be determined by flow cytometry as described above as determined by STAT (e.g., STAT1, STAT3, STAT5) phosphorylation levels according to methods well known in the art.

[0079] Significantly reduced binding: As used herein, the term "exhibiting significantly reduced binding" refers to a variant of a first molecule (e.g., a ligand or antibody) that exhibits significantly reduced affinity for a second molecule (e.g., a receptor or antigen) compared to the parent form of the first molecule. With respect to antibody variants, an antibody variant "exhibits significantly reduced binding" if it binds to the native receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the parent antibody from which the variant is derived. Similarly, with respect to variant ligands, a variant ligand "exhibits significantly reduced binding" if it binds to the receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the parent ligand from which the variant ligand is derived. Similarly, with respect to a variant receptor, a variant ligand "exhibits significantly reduced binding" if the affinity of the variant receptor binds with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the parent receptor from which the variant receptor is derived.

[0080] Small molecules: The term "small molecule" refers to a compound (typically a pharmaceutically active compound) having a molecular weight of less than about 10 kDa, less than about 2 kDa, or less than about 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic molecules, molecules containing radioactive atoms, and synthetic molecules. The term "small molecule" is a term well understood by those skilled in the art of pharmaceutical sciences and is typically used to distinguish organic compounds from biologics.

[0081] Specific binding to: As used herein, the term "specifically bind" refers to the degree of affinity a first molecule exhibits for a second molecule. In the context of binding pairs (e.g., ligand / receptor, antibody / antigen), a first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the first molecule does not bind in significant amounts to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the affinity of the first molecule for the second molecule is at least 2-fold, alternatively at least 5-fold, alternatively at least 10-fold, alternatively at least 20-fold, or alternatively at least 100-fold greater than the affinity of the first molecule for other components present in the sample. In certain embodiments, when the first molecule of a binding pair is an antibody, the equilibrium dissociation constant between the antibody and the antigen is about 10, as 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 M is greater than M, the antibody specifically binds to the antigen (or antigenic determinant (epitope) of a protein, antigen, ligand, or receptor). In one aspect, when the ligand is an IL27Rα-binding sdAb and the receptor comprises IL27Rα, the equilibrium dissociation constant of IL27Rα-binding sdAb / IL27RαECD is greater than or equal to about 10 5 Over M or about 10 6 Over M or about 10 7Over M or about 10 8 Over M or about 10 9 Over M or about 10 10 More than M or about 10 11If it is greater than M, the IL27Rα-binding sdAb binds specifically. Specific binding can be assessed using techniques known in the art, including, but not limited to, competitive ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plots, non-linear curve-fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET)); solution-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation); and solid-phase ligand binding assays (e.g., multiwell plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)), and surface plasmon resonance assays (see, e.g., Drescher et al., (2009) Methods Mol Biol 493:323-343 and commercially available instrumentation, e.g., Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA 01752)). In some embodiments, the present disclosure provides molecules that specifically bind to IL27Rα isoforms (e.g., IL27Rα-binding sdAbs). As used herein, the binding affinity of an IL27Rα-binding molecule to IL27Rα, binding affinity may be determined and / or quantified by surface plasmon resonance ("SPR"). When assessing the binding affinity of an IL27Rα-binding molecule to IL27Rα, one member of the binding pair may be immobilized and the other member of the binding pair may be provided in the mobile phase.In some embodiments, the sensor chip on which the protein of interest is immobilized is conjugated with a substance that facilitates binding of the protein of interest, such as a nitrilotriacetic acid (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., an 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 to the chip surface to assess binding. For example, a polyhistidine sequence (e.g., 6xHis) is added for retention on the chip. (SEQ ID NO:195) or 8xHis (SEQ ID NO:196) One member of the binding pair to be evaluated was conjugated by NTA by incorporating a chelating peptide containing the IL27Rα-binding molecule. In some embodiments, the IL27Rα-binding molecule may be immobilized on a chip, and IL27Rα (or its ECD fragment) may be provided in the mobile phase. Alternatively, IL27Rα (or its ECD fragment) may be immobilized on a chip, and the IL27Rα-binding molecule may be provided in the mobile phase. In either case, it should be noted that modifying some proteins for immobilization on a coated SPR chip may interfere with the binding properties of one or both components of the binding pair to be evaluated by SPR. In such cases, it may be necessary to switch the mobile and binding members of the binding pair, or to use a chip with a binder that facilitates non-interfering conjugation of the protein to be evaluated. Alternatively, when using SPR to evaluate the binding affinity of an IL27Rα-binding molecule to IL27Rα, the IL2Rb-binding molecule may be conjugated to a poly-His sequence (e.g., 6xHis). (SEQ ID NO:195) or 8xHis (SEQ ID NO:196)The IL27Rα receptor subunit may be derivatized by the C-terminal addition of a poly-His sequence and immobilized on an NTA-derivatized sensor chip, with the IL27Rα receptor subunit for which ligand binding affinity is being evaluated being provided in the mobile phase. Means for incorporating a poly-His sequence at the C-terminus of an IL27Rα-binding molecule produced by recombinant DNA techniques are well known to those skilled in the relevant fields of biotechnology. In some embodiments, the binding affinity of the IL27Rα-binding molecule to IL27Rα is measured using SPR generally in accordance with the disclosure of the Examples.

[0082] subject: The terms "recipient," "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some aspects, the mammal is a human.

[0083] Virtually pure: As used herein, the term "substantially pure" indicates that a component of a composition constitutes more than about 50%, alternatively more than about 60%, alternatively more than about 70%, alternatively more than about 80%, alternatively more than about 90%, or alternatively more than about 95% of the total content of the composition. A "substantially pure" protein constitutes more than about 50%, alternatively more than about 60%, alternatively more than about 70%, alternatively more than about 80%, alternatively more than about 90%, or alternatively more than about 95% of the total content of the composition.

[0084] T cells: As used herein, the term "T cell" ("T-cell" or "T cell") is used in its conventional sense to refer to lymphocytes that differentiate in the thymus, have specific cell surface antigen receptors, and control the initiation or suppression of cellular and humoral immunity, including those 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, e.g., 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, recombinantly modified TILs, and TCR-engineered cells. In some embodiments, the T cells are T cells that express the IL27Rα isoform, which are referred to interchangeably as IL27Rα cells, IL27Rα+ cells, IL27Rα T cells, or IL27Rα+ T cells.

[0085] Terminus / Terminal: As used herein in the context of a polypeptide structure, the terms "N-terminus" (or "amino-terminus") and "C-terminus" (or "carboxyl-terminus") refer to the extreme amino- and carboxyl-termini of a polypeptide, respectively. In contrast, the terms "N-terminus" and "C-terminus" refer to relative positions in a polypeptide amino acid sequence relative to the N-terminus and C-terminus, respectively, and may include residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being proximal to the N-terminus of the polypeptide. "Immediately C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being proximal to the C-terminus of the polypeptide.

[0086] Transmembrane domain: The term "transmembrane domain" or "TM" refers to a polypeptide domain of a transmembrane polypeptide (e.g., a transmembrane receptor) that is embedded in the cell membrane and peptidyl-linked to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide when the transmembrane polypeptide is associated with the cell membrane. The transmembrane domain may be homologous (naturally associated) or heterologous (not naturally associated) with either or both of the extracellular domain and / or intracellular domain. In some embodiments, when a receptor is a chimeric receptor that includes an intracellular domain derived from a first parent receptor and a second extracellular domain derived from a second, different parent receptor, the transmembrane domain of the chimeric receptor is the transmembrane domain normally associated with either the ICD or ECD of the parent receptor from which the chimeric receptor is derived.

[0087] To treat: The terms "treat," "treating," "treatment," and the like refer to a course of action (e.g., contacting a subject with a pharmaceutical composition comprising an IL27Rα-binding sdAb alone or in combination with adjuvants) undertaken with a subject in response to a diagnosis that the subject is suffering from a disease, disorder, or condition, or a symptom thereof, wherein the course of action is undertaken to temporarily or permanently eliminate, reduce, inhibit, alleviate, or ameliorate at least one of: (a) the underlying cause of such disease, disorder, or condition afflicting the subject; and / or (b) at least one symptom associated with such disease, disorder, or condition. In some embodiments, treating includes a course of action taken with respect to a subject suffering from a disease, wherein the course of action inhibits the disease in the subject (e.g., prevents the development of, or ameliorates, one or more symptoms associated with, the disease, disorder, or condition).

[0088] Treg cells or regulatory T cells: The terms "regulatory T cells," "Treg cells," or "Treg" refer to effector T cells (T eff ) that can suppress the responses of other T cells, including but not limited to CD4+ The terms "conventional CD4+ T cells" and "conventional CD4+ T cells" are used interchangeably herein to refer to a type of T cell. Treg cells are typically characterized by 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 cells" refers to non-Treg CD4 + T cells to CD4 + Used to distinguish it from Treg.

[0089] Variants: The terms "variant," "protein variant," or "variant protein," or "variant polypeptide" are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide by at least one amino acid modification, substitution, or deletion. The parent polypeptide can be a native or wild-type (WT) polypeptide or a modified version of a WT polypeptide. The term variant polypeptide can refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding it. In some embodiments, a variant polypeptide contains about 1 to about 10, alternatively about 1 to about 8, alternatively about 1 to about 7, alternatively about 1 to about 5, alternatively about 1 to about 4, alternatively about 1 to about 3, alternatively about 1 to 2 amino acid modifications, substitutions, or deletions, or a single amino acid modification, substitution, or deletion, relative to the parent polypeptide from which the variant is derived. A variant may be at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 95% identical, or alternatively at least about 90% identical to the parent polypeptide from which the variant is derived.

[0090] Wild type: As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A wild-type protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been modified by the hand of man.

[0091] IL27Rα The IL27Rα binding molecules of the present disclosure specifically bind to the extracellular domain of IL27Rα.

[0092] Human IL27Rα In one embodiment, it specifically binds to the extracellular domain of the human IL27Rα receptor subunit (hIL27Rα). hIL27Rα is expressed as a 636 amino acid precursor that contains a 32 amino acid N-terminal signal sequence that is cleaved post-translationally to yield the 604 amino acid mature protein. The classical full-length hIL27Rα precursor (including the signal peptide) has the amino acid sequence: It is a 636 amino acid polypeptide with the sequence TIFF0007652883000022.tif62134.

[0093] For purposes of this disclosure, the numbering of amino acid residues in the human IL27Rα polypeptide as described herein is based on the numbering of this classical sequence (UniProt Reference No. Q6UWB1, SEQ ID NO:1). Amino acids 1-32 of SEQ ID NO:1 are identified as the signal peptide of hIL27Rα, amino acids 33-516 of SEQ ID NO:1 are identified as the extracellular domain, amino acids 517-537 of SEQ ID NO:1 are identified as the transmembrane domain, and amino acids 538-636 of SEQ ID NO:1 are identified as the intracellular domain.

[0094] For the purpose of generating antibodies that bind to the ECD of IL27Rα, immunization can be carried out using the extracellular domain of hIL27Rα. The extracellular domain of hIL27Rα has the sequence: It is a 484 amino acid polypeptide of TIFF0007652883000023.tif49134.

[0095] Mouse IL27Rα In one embodiment, it specifically binds to the extracellular domain of the mouse or murine IL27Rα receptor subunit (mIL27Rα). mIL27Rα is expressed as a 623 amino acid precursor that contains a 24 amino acid N-terminal signal sequence that is cleaved post-translationally to yield a 599 amino acid mature protein. The classical full-length mIL27Rα precursor (including the 24 amino acid signal peptide) has the amino acid sequence: It is a 623 amino acid polypeptide with the sequence TIFF0007652883000024.tif62134.

[0096] For purposes of this disclosure, the numbering of amino acid residues in the mIL27Rα polypeptide as described herein is based on the numbering of this classical sequence (UniProt Reference No. O70394, SEQ ID NO:193). Amino acids 1-24 of SEQ ID NO:193 are identified as the signal peptide of mIL27Rα, amino acids 23-510 of SEQ ID NO:193 are identified as the extracellular domain, amino acids 511-531 of SEQ ID NO:193 are identified as the transmembrane domain, and amino acids 532-623 of SEQ ID NO:193 are identified as the intracellular domain.

[0097] Immunization can be performed with the extracellular domain of mIL27Rα to generate antibodies that bind to the ECD of IL27Rα. The extracellular domain of the mIL27Rα receptor has the sequence: It is a 486 amino acid polypeptide of TIFF0007652883000025.tif49133.

[0098] IL27Rα binding molecules and single domain antibodies In some aspects, the IL27Rα-binding molecules of the present disclosure are single domain antibodies (sdAbs). The present disclosure relates to IL27Rα-binding molecules, including single domain antibodies (sdAbs), that specifically bind to the extracellular domain of the human IL27Rα isoform (hIL27Rα), which is found on all IL27Rα-expressing cells.

[0099] Single domain antibodies (sdAbs) are antibodies containing one monomeric variable antibody domain. Like full-length antibodies, sdAbs can specifically bind to an antigenic determinant. hIL27Rα-binding VHH single domain antibodies can be engineered from heavy chain antibodies isolated from camelids (e.g., camels, llamas, dromedaries, alpacas, and guanacos) immunized with the extracellular domain of hIL27Rα or an immunologically active fragment thereof. Descriptions of sdAbs and VHHs are found, for example, in De Greve et al., (2019) Curr Opin Biotechnol. 61:96-101; Ciccarese, et al., (2019) Front Genet. 10:997; Chanier and Chames (2019). Antibodies (Basel) 8(1); and De Vlieger, et al. (2018) Antibodies(Basel) 8(1). Alternatively, hIL27Rα single-domain antibodies may be engineered from heavy chain antibodies isolated from IgNAR heavy chain antibodies isolated from cartilaginous fish immunized with the extracellular domain of hIL27Rα or an immunologically active fragment thereof. hIL27Rα-binding sdAbs may also be obtained by splitting dimeric variable domains derived from immunoglobulin G (IgG) isotypes from other mammalian species, including humans, rats, and rabbits, immunized with the extracellular domain of hIL27Rα or an immunologically active fragment thereof. Currently, most sdAb research is based on the heavy chain variable domain, but sdAbs derived from the light chain have also been shown to specifically bind to target proteins containing antigenic immunization sequences. Moller et al., J Biol Chem. 285(49):38348-38361, 2010.

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

[0101] The present disclosure provides IL27Rα binding molecules comprising a polypeptide having at least 75%, alternatively 80%, alternatively 90%, alternatively 95%, alternatively 98%, alternatively 99%, or alternatively 100% identity to the polypeptide of any one of SEQ ID NOs:2-25.

[0102] The present disclosure provides IL27Rα binding molecules comprising a polypeptide having at least 75%, alternatively 80%, alternatively 90%, alternatively 95%, alternatively 98%, alternatively 99%, or alternatively 100% identity to the polypeptide of any one of SEQ ID NOs:61-74.

[0103] The present disclosure provides IL27Rα binding molecules comprising CDR1, CDR2, and CDR3 as set forth in a row of Table 1 provided herein. In some embodiments, CDR1, CDR2, and CDR3 may each independently comprise at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence set forth in a row of Table 1 provided herein, and may have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes.

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

[0105] experiment The single-domain antibodies of the present disclosure were obtained from camels by immunization with the extracellular domain of the IL27Rα receptor. The IL27Rα VHH molecules of the present disclosure were generated generally according to the disclosure in the Examples. Briefly, camels were immunized over several weeks with subcutaneous adjuvanted compositions containing recombinantly produced fusion proteins comprising the extracellular domain of IL27Rα, a human IgG1 hinge domain, and a human IgG1 heavy chain Fc, using the ECDs of human IL27Rα and mouse IL27Rα. After immunization, RNA extracted from blood samples of appropriately sized VHH-hinge-CH2-CH3 species was transcribed to generate DNA sequences, digested, and an approximately 400-bp fragment containing the nucleic acid sequence encoding the VHH domain was identified and isolated. To facilitate insertion into a phagemid vector in frame with the sequence encoding the his-tag, the isolated sequences were digested with restriction endonucleases and transformed into Escherichia coli (E. coli) to generate a phage library. Multiple rounds of phage library biopanning were performed to identify VHHs that bound to the ECD of IL27Rα (human or mouse, as appropriate). Individual phage clones were isolated for selective binding, confirmed by periplasmic extract ELISA (PE-ELISA) in a 96-well plate format and colorimetric assay. IL27Rα-binding molecules that showed specific binding to the IL27Rα antigen were isolated and sequenced and analyzed to identify VHH sequences, CDRs, and unique VHH clonotypes. As used herein, the term "clonotype" refers to a collection of binding molecules generated from the same B-cell precursor within a specific collection of antigen-binding molecules belonging to the same germline family, having the same CDR3 length, and sharing 70% or more homology in CDR3 sequence. VHH molecules (anti-human IL27Rα VHHs) that showed specific binding to the hIL27Rα ECD antigen and the CDRs isolated from these VHHs are shown in Table 1. VHH molecules (anti-mouse IL27Rα VHHs) that showed specific binding to the mIL27Rα ECD antigen and the CDRs isolated from these VHHs are shown in Table 3.The nucleic acid sequences encoding the VHHs in Tables 1 and 3 are shown in Tables 2 and 4, respectively.

[0106] To more fully characterize the binding properties and assess the binding affinity of the VHH molecules generated as described above, representatives of each human VHH clonotype were subjected to analysis by surface plasmon resonance, generally as disclosed herein in Example 5. The results of these SPR studies are summarized in Table 6 below.

[0107] Table 6: Binding of anti-hIL27Ra monoFc VHH (ligand) to hIL27Ra-his (Antigen: Origene, Catalog No. TP307012) TIFF0007652883000026.tif54168 * Both the association and dissociation rate constants may be depressed at Rmax > 100. If this effect exists, it is likely to be offset by the reaction rate ratio, i.e., the affinity constant.

[0108] As evidenced by the data presented in Table 6 above, the IL27Rα VHH binding molecules exhibited specific binding to the antigen and displayed a range of affinities for the IL27Rα antigen.

[0109] In some cases, due to sequence or structural similarity between the extracellular domains of IL27Rα receptors from various mammalian species, immunization with an antigen derived from IL27Rα of a first mammalian species (e.g., hIL27Rα-ECD) may produce antibodies that specifically bind to the IL27Rα receptor of one or more additional mammalian species. Such antibodies are said to be "cross-reactive." For example, immunization of a camelid with a human-derived antigen (e.g., hIL27Rα-ECD) may produce antibodies that are cross-reactive with the mouse receptor and the human receptor. Assessment of the cross-reactivity of antibodies to receptors from other mammalian species can be readily confirmed by one of skill in the art using, for example, methods related to assessing binding affinity and / or specific binding described elsewhere herein, such as flow cytometry or SPR. Consequently, the use of the term "human IL27Rα VHH" or "hIL27Rα VHH" merely indicates that the species of IL27Rα antigen used to immunize the camelid from which the VHH originated was human IL27Rα (e.g., hIL27Rα, ECD, SEQ ID NO:192), and should not be construed as a limitation on the specific binding affinity of the VHH for hIL27Rα molecules of other mammalian species. Similarly, the use of the term "mouse IL27Rα VHH" or "mIL27Rα" merely indicates that the species of IL27Rα antigen used to immunize the camelid from which the VHH originated was mouse IL27Rα (e.g., mIL27Rα ECD, SEQ ID NO:194), and should not be construed as a limitation on the specific binding affinity of the VHH for IL27Rα molecules of other mammalian species.

[0110] Engineered single domain antibodies CDR-grafted sdAb In some embodiments, the IL27Rα-binding sdAb of the present disclosure is a CDR-grafted IL27Rα-binding sdAb. To generate a CDR-grafted sdAb, CDRs obtained from an antibody, a heavy chain antibody, and an sdAb derived therefrom may be grafted onto another framework as described in Saerens, et al. (2005) J. Mol Biol 352:597-607. In some embodiments, the present disclosure provides an IL27Rα-binding molecule comprising a CDR-grafted IL27Rα-binding sdAb, wherein the CDR-grafted IL27Rα-binding sdAb comprises a set of CDR1, 2, and 3 as shown in the rows of Table 3 above.

[0111] Chimeric and humanized sdAbs Any framework region can be used with the CDRs as described herein. In some embodiments, the IL27Rα-binding sdAb is a chimeric sdAb, in which the CDRs are derived from one species (e.g., camel) and the framework and / or constant regions are derived from another species (e.g., human or mouse). In certain embodiments, the framework regions are human or humanized sequences. Thus, a humanized IL27Rα-binding sdAb derived from a hIL27Rα-binding VHH is considered within the scope of the present disclosure. Techniques for humanizing camelid single-domain antibodies are well known in the art. See, for example, Vincke, et al. (2009) General Strategy to Humanize a Camelid Single-Domain Antibody and Identification of a Universal Humanized Nanobody Scaffold J. Biol. Chem. 284(5)3273-3284.

[0112] In some embodiments, the V H H can be humanized to contain human framework regions. HExamples of human germlines that can be used to generate H include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).

[0113] IL27Rα-binding molecules comprising an additional agent In some embodiments, the IL27Rα-binding molecules of the present disclosure comprise an IL27Rα single domain antibody (sdAb) conjugated to one or more additional biologically active agents, including, but not limited to, a therapeutic agent, a chemically active agent, an optically active agent, or a radioactive agent, including combinations of a therapeutic agent, a chemically active agent, an optically active agent, or a radioactive agent. The conjugation of at least one such biologically active agent, a chemically active agent, an optically active agent, or a radioactive agent confers additional biological or chemical properties to the IL27Rα-binding sdAb, and this combination results in an IL27Rα-binding molecule with additional or different utilities.

[0114] For example, the additional agent may be a molecule selected from one or more of the following: an immunomodulator (e.g., an immunogen); a molecule that improves water solubility (e.g., water-soluble polymers and hydrophilic molecules, e.g., sugars); a carrier molecule that increases in vivo half-life (e.g., PEGylation, Fc fusion, or acylation); generation of antibodies for use in detection assays (e.g., epitope tags) or to increase ease of purification (e.g., chelating peptides, e.g., poly-His tags); a targeting domain that selectively targets the IL27Rα-binding molecule to a specific cell or tissue type; a therapeutic agent (e.g., a therapeutic agent comprising a small molecule or polypeptide agent); or an agent that enhances visibility to optical or electromagnetic sensors (e.g., a radionucleotide or fluorescent substance). In some embodiments, the linker is a cleavable or non-cleavable linker. As contemplated herein, the use of a cleavable linker in an IL27Rα-binding molecule facilitates release of the therapeutic agent into the intracellular cytoplasm upon internalization of the IL27Rα-binding molecule. The use of a non-cleavable linker would allow for release of the IL27Rα binding molecule upon digestion, or the non-cleavable linker could be used with an agent that does not require release from the antibody (e.g., an imaging agent).

[0115] In some embodiments, the IL27Rα-binding molecule comprises an IL27Rα-binding sdAb stably linked to an additional agent via a linker. The linker is a covalent bond between two elements of the IL27Rα-binding molecule (e.g., a hIL27Rα-binding VHH and a PEG polymer). The linker may be a covalent bond, a chemical linker, or a peptide linker. Suitable linkers generally include a "flexible linker" of sufficient length to allow some movement between the IL27Rα-binding sdAb and the linked agent. Examples of chemical linkers include aryl acetylene, 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. Suitable peptide linkers can be readily selected and can be of any suitable length, for example, a peptide linker of 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of flexible linkers include glycine polymers (Gly). n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers are relatively structurally undefined and therefore may serve as neutral tethers between components. Further examples of flexible linkers include glycine polymers (G), n, glycine-alanine polymers, alanine-serine polymers, and glycine-serine polymers. Glycine and glycine-serine polymers are relatively structurally undefined and, therefore, may serve as neutral tethers between components. Multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of such linker sequences may be linked together to provide flexible linkers that can be used to conjugate heterologous amino acid sequences to the IL27Rα-binding sdAbs disclosed herein. In some embodiments, the linker has the formula (GGGS)n (SEQ ID NO:197) , (GGGSG)n (SEQ ID NO:198) , (GGGGS)n (SEQ ID NO:199) , (GGS)nG (SEQ ID NO:200) , or (GGSG)n (SEQ ID NO:201) wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0116] immunomodulators In some embodiments, the IL27Rα-binding molecules of the present disclosure are operably linked to immunomodulators (immunoconjugates). Immunomodulators that can be conjugated to the hIL27Rα-binding sdAbs of the present disclosure include, but are not limited to, inactivated virus particles, inactivated bacterial toxins, such as toxoids from diphtheria, tetanus, or cholera, or leukotoxin molecules, inactivated bacteria, and dendritic cells. Such immunoconjugates are useful in promoting immune responses against IL27Rα or cells expressing IL27Rα.

[0117] Flag tag In some embodiments, the IL27Ra-binding molecules of the present disclosure are operably linked to an antigenic tag, such as a FLAG sequence, which 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 IL27Ra-binding sdAb polypeptide further comprises a C-terminal c-myc epitope tag.

[0118] Chelating Peptides In some embodiments, the IL27Rα-binding molecules of the present disclosure are operably linked to one or more transition metal chelating polypeptide sequences. Incorporation of such a transition metal chelating domain facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Patent No. 4,569,794 to Smith et al., issued February 11, 1986. Examples of transition metal chelating polypeptides useful in the practice of the present IL27Rα-binding molecules are described in Smith et al., supra, and U.S. Patent No. 5,320,663 to Dobeli et al., issued May 10, 1995, the entire disclosures of which are incorporated herein by reference. Particular transition metal chelating polypeptides useful in the practice of the present IL27Rα-binding molecules contain a sequence of three to six consecutive histidine residues. (SEQ ID NO:202) Polypeptides containing, for example, 6 histidine (His) 6 peptides (SEQ ID NO:195)and is often referred to in the art as a "His tag." In addition to providing a purification "handle" for recombinant proteins or for facilitating immobilization on an SPR sensor chip, conjugation of hIL27Rα-binding molecules to such chelating peptides facilitates targeted delivery of transition metal ions to IL27Rα-expressing cells as kinetically inert or kinetically unstable complexes, generally 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 transition metal ion can be a reporter molecule, e.g., a fluorescent compound or a radioimaging agent, including a radioactive substance or a therapeutic agent.

[0119] Carrier molecules In some embodiments, the IL27Rα-binding sdAbs of the present disclosure may be conjugated to one or more carrier molecules. Carrier molecules are typically large, slowly metabolized macromolecules that provide in vivo stability and / or a long duration of action, distinguishing such molecules from traditional carrier molecules used in the preparation of pharmaceutical formulations, as described below. Examples of in vivo carriers that can be incorporated into IL27Rα-binding molecules include, but are not limited to, proteins (including, but not limited to, human serum albumin); fatty acids (acylated); polysaccharides (including, but not limited to, (N-linked and O-linked) sugars, Sepharose, agarose, cellulose, or cellulose); polypeptide amino acid copolymers; acylated, or polysialylated, polyethylene glycol (PEG) polymers.

[0120] Water-soluble polymers In some embodiments, the IL27Rα-binding sdAb is conjugated to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present IL27Rα-binding molecules include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefin alcohols, polysaccharides, polyα-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0121] Polyethylene glycol In one embodiment, the carrier molecule is a polyethylene glycol ("PEG") polymer. Conjugation of PEG polymers to proteins (PEGylation) is a well-established method for extending the serum half-life of biological agents. Furthermore, PEGylated polypeptides may be referred to as mono-PEGylated, di-PEGylated, tri-monoPEGylated (etc.) to refer to polypeptides having one, two, three (or more) PEG moieties, respectively, attached to the polypeptide. In some embodiments, PEG may be covalently attached directly to the sdAb (e.g., via a lysine side chain, a sulfhydryl group of cysteine, or an N-terminal amine), optionally using a linker between the PEG and the sdAb. In some embodiments, the IL27Rα binding molecule comprises multiple PEG molecules, each of which is attached to a different amino acid residue. In some embodiments, the sdAb may be modified by incorporating unnatural amino acids with unnatural amino acid side chains to facilitate site-specific PEGylation. In other embodiments, cysteine ​​residues at one or more positions in the sdAb may be substituted to facilitate site-specific PEGylation via cysteine ​​sulfhydryl side chains.

[0122] In some cases, the IL27Rα-binding molecules of the present disclosure have an N-terminal glutamine ("1Q") residue. N-terminal glutamine residues have been observed to spontaneously cyclize to form pyroglutamate (pE) under or near physiological conditions (see, e.g., Liu, et al. (2011) J. Biol. Chem. 286(13): 11211-11217). In some embodiments, the formation of pyroglutamate complicates N-terminal PEG conjugation, particularly when aldehyde chemistry is used for N-terminal PEGylation. Consequently, when PEGylating the IL27Rα-binding molecules of the present disclosure, particularly when aldehyde chemistry is used, position 1 of an IL27Rα-binding molecule having an amino acid at position 1 (e.g., 1Q) is replaced with an alternative amino acid or position 1 is deleted (e.g., des-1Q). In some embodiments, an IL27Rα binding molecule of the disclosure comprises an amino acid substitution selected from the group of Q1E and Q1D.

[0123] PEG suitable for conjugation to polypeptide sequences is generally water-soluble at room temperature and has the general formula: R(O-CH2-CH2) n OR where R is hydrogen or a protecting group, e.g., an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has 1 to 8 carbons. PEG can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.

[0124] The molecular weight of PEG used in IL27Rα-binding molecules is not limited to any particular range. The molecular weight of the PEG component of an IL27Rα-binding molecule may be greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. a linear or branched PEG molecule having a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, alternatively about 30,000 to about 40,000 daltons. In one embodiment of the IL27Rα-binding molecule, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0125] The present disclosure also contemplates IL27Rα-binding molecules containing multiple PEG moieties, where the PEGs have different size values ​​and, therefore, the various different PEGs are present in specific ratios. For example, in preparing PEGylated IL27Rα-binding molecules, some compositions contain a mixture of mono-, di-, tri-, and quadra-PEGylated sdAb conjugates. In some compositions, the percentage of mono-PEGylated species is 18-25%, the percentage of di-PEGylated species is 50-66%, the percentage of tri-PEGylated species is 12-16%, and the percentage of quadra-PEGylated species is up to 5%. Such complex compositions can be produced using reaction conditions and purification methods known in the art. Chromatography can be used to separate conjugate fractions, and then, for example, fractions containing conjugates with a desired number of PEGs attached can be identified and purified from unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0126] PEGylation is most frequently performed at the α-amino group at the N-terminus of a polypeptide, the ε-amino group in the side chain of a lysine residue, and the imidazole group in the side chain of a histidine residue. Because most recombinant polypeptides have one α-amino group and multiple ε-amino and imidazole groups, a large number of positional isomers can be generated depending on the linker chemistry.

[0127] Two widely used first-generation activated monomethoxy PEGs (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. Pat. No. 5,650,234), which react preferentially 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 a polypeptide via reductive amination.

[0128] PEG can be attached to the IL27Rα-binding molecules of the present disclosure via a terminal reactive group ("spacer") that mediates a bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEGs having a spacer that can be attached to a free amino group include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0129] In some embodiments, PEGylation of sdAbs is facilitated by incorporating unnatural amino acids with unique side chains to facilitate site-specific PEGylation. To achieve site-specific PEGylation of such polypeptides, the incorporation of unnatural amino acids into polypeptides to provide functional moieties is known in the art. See, for example, Ptacin, et al., PCT International Application No. PCT / US2018 / 045257, filed August 3, 2018, and published February 7, 2019, as International Publication No. WO2019 / 028419A1.

[0130] The PEG moiety of a PEGylated IL27Rα binding molecule can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.Specific embodiments of PEG useful in the practice of the present disclosure include 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (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 containing two 20kDA linear PEG molecules Included are PEG-NHS ester (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL), and linear 30 kDa PEG-NHS ester.

[0131] Fc fusion In some embodiments, the carrier molecule is an Fc molecule or a monomeric subunit thereof. In some embodiments, the dimeric Fc molecule may be engineered to have "knob-into-hole modifications." Knob-into-hole modifications are more fully described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621, as well as 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. A knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, in which i) an amino acid residue in the CH3 domain of a first heavy chain is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) to create a protruding portion ("knob") from the surface, or ii) an amino acid residue in the CH3 domain of a 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") within the interface at the second CH3 domain, and the protruding side chain ("knob") of the first CH3 domain is accommodated in the cavity at the second CH3 domain. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W, and optionally 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. Additionally, the Fc domain may be modified by introducing a cysteine ​​residue at position S354 in one chain and Y349 in the other chain, resulting in a stabilizing disulfide bond between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15).The knobs-into-holes format is used to facilitate expression of a first polypeptide (e.g., an IL27Rα-binding sdAb) on a first Fc monomer that has a "knob" modification to facilitate expression of a heterodimeric polypeptide conjugate, and a second polypeptide on a second Fc monomer that has a "hole" modification.

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

[0133] In some embodiments of the IL27Rα-binding molecule, the IL27Rα-binding molecule can be targeted to a specific cell type by incorporating a targeting domain into the structure of the IL27Rα-binding molecule. As used herein, the term targeting domain refers to a moiety that specifically binds to a molecule expressed on the surface of a target cell. The targeting domain can be any moiety that specifically binds to one or more cell surface molecules (e.g., T cell receptors) expressed on the surface of the target cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is an IL27Rα+ T cell.

[0134] In some embodiments, the targeting domain is a ligand for a receptor. In some embodiments, the targeting domain is a ligand for a receptor expressed on the surface of a T cell. In some embodiments, the ligand is a cytokine. In some embodiments, the cytokine includes, but is not limited to, the group consisting of interleukins, interferons, and functional derivatives thereof. In some embodiments, the cytokine includes, but is not limited to, the group consisting of IL2, IL3, IL4, IL7, IL9, IL12, IL15, IL18, IL21, IL22, IL23, IL27, IL28, IL34, and modified versions or fragments thereof that bind to their cognate ligands expressed on the surface of T cells. In some embodiments, the cytokine includes, but is not limited to, the group consisting of interferon alpha, interferon a2b, interferon gamma, or interferon lambda, and modified versions or fragments thereof that bind to their cognate ligands expressed on the surface of T cells.

[0135] In another aspect, the present disclosure provides a multivalent binding molecule comprising (a) an IL27Rα-binding molecule and (b) a second binding molecule that specifically binds to the extracellular domain of a second cell surface molecule, wherein the IL27Rα-binding molecule and the second binding molecule are operably linked, optionally via a chemical or polypeptide linker. In some embodiments, the IL27Rα-binding molecules of the present disclosure are useful in preparing the multivalent binding molecules described in Gonzalez, et al. PCT / US2018 / 021301, published as WO2018 / 182935A1 on October 4, 2018. In some aspects, the second binding molecule specifically binds to the extracellular domain of (i) a component of a cytokine receptor that activates the JAK / STAT pathway in a cell; (ii) a receptor tyrosine kinase; or (iii) 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, 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 (TNFRSF3), OX40 (TNFRSF4), RANK (TNFRSF11A), TACI (TNFRSF13B), Troy (TNFRSF19), XEDAR (TNFRSF27), osteoprotegerin (TNFRSF11B), TWEAK receptor (TNFRSF12A), BAFF receptor (TNFRSF13C), and NGF receptor (TNFRSF16) are TNFR superfamily members.

[0136] In some embodiments, the targeting domain is a polypeptide that specifically binds to a cell surface molecule associated with a tumor cell (e.g., a cognate ligand of a tumor cell receptor) selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Ra2, CD19, mesothelin, Her2, EpCam, Mucl, ROR1, CD133, CEA, EGFRRVIII, PSCA, GPC3, Pan-ErbB, and FAP.

[0137] In some embodiments, the targeting domain of the IL27Ra-binding molecule is an antibody (as defined above to include molecules such as VHHs, scFvs, etc.). Examples of antibodies that can be incorporated as the targeting domain of an IL27Ra-binding molecule include, but are not limited to, the group consisting of anti-GD2, anti-BCMA, anti-CD19, anti-CD33, anti-CD38, anti-CD70, anti-GD2 and IL3Ra2 antibodies, anti-CD19, anti-mesothelin, anti-Her2, anti-EpCam, anti-Muc1, anti-ROR1, anti-CD133, anti-CEA, anti-PSMA, anti-EGFRRVIII, anti-PSCA, anti-GPC3, anti-Pan-ErbB, and anti-FAP antibodies.

[0138] The antibody or antigen-binding fragment thereof can be linked to another antibody to form, for example, a bispecific or multispecific antibody.

[0139] sign In some embodiments, the IL27Rα-binding molecules of the present disclosure are operably linked to a label. In some embodiments, the label is incorporated to facilitate use as an imaging agent, a diagnostic agent, or for use in cell sorting procedures. The term label includes, but is not limited to, a fluorescent label, a biologically active enzyme label, a radioisotope (e.g., a radioactive ion), a nuclear magnetic resonance active label, a luminescent label, or a magnetic compound. In one embodiment, the IL27Rα-binding sdAb (e.g., an IL27Rα-binding VHH) molecule is stably associated (e.g., covalently bound, coordinately covalently bound) with an imaging label. The term imaging label is used to describe any of a variety of compounds that are signatures that facilitate identification, tracking, and / or location of the IL27Rα-binding sdAb (or its metabolites) using diagnostic procedures. Examples of imaging labels include, but are not limited to, fluorescent compounds, radioactive compounds, and compounds that are opaque to imaging methods (e.g., X-rays, ultrasound). Examples of radioactive compounds useful as imaging labels 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 Rh), but are not limited to:

[0140] therapeutic agent In some embodiments, the IL27Rα binding molecules of the present disclosure are operably linked to a therapeutic agent. Examples of therapeutic agents include antibodies, cytotoxic or cytostatic compounds, radioisotopes, plant-, fungal-, or bacterial-derived molecules, or biological proteins (e.g., protein toxins) or particles (e.g., nanoparticles or recombinant viral particles, e.g., recombinant viral particles via viral coat proteins), therapeutic antibodies, therapeutic small molecules (e.g., chemotherapeutic agents), or biotherapeutics, including chemotherapeutic agents, as described more fully herein.

[0141] In some embodiments, therapeutic agents that can be incorporated into the IL27Rα-binding molecules of the present disclosure are short-range radiation emitters, including, for example, short-range, high-energy α-emitters. Examples of such radioisotopes include α-emitters, β-emitters, γ-emitters, or β / γ-emitters. Radioisotopes useful as therapeutic agents include yttrium-90 ( 90 Y), lutetium-177( 177 Lu), Actinium-225( 225 Ac), astatine-211( 211 At), Rhenium-186( 186 Re), Bismuth-212( 212 Bi), Bismuth-213( 213Bi), and rhodium-188( 188 Rh) is included.

[0142] In some embodiments, an IL27Rα binding molecule of the disclosure is operably linked to a cytotoxic agent (or derivative thereof), such as maytansinol or DM1 maytansinoid), a taxane, or calicheamicin, Pseudomonas exotoxin A, devuganin, ricin toxin, diphtheria toxin, an amatoxin, such as α-amanitin, saporin, maytansine, maytansinoid, auristatin, an anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer, or variants thereof.

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

[0144] chemical synthesis In addition to producing mutant polypeptides through the expression of nucleic acid molecules altered by recombinant molecular biology techniques, the polypeptide domain of an IL27Ra-binding molecule 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 a polypeptide domain of an IL27Ra-binding molecule exhibiting the described properties. This method can incorporate natural and unnatural amino acids at desired positions to facilitate the attachment of a particular molecule (e.g., PEG).

[0145] In some embodiments, the polypeptide domain of an IL27Rα-binding molecule of the present disclosure can be produced by chemical synthesis. Chemical synthesis of the polypeptide domain of an IL27Rα-binding molecule can proceed via solution phase or solid phase. Solid-phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and / or peptide / protein backbone modifications. Various types of SPPS can be used to synthesize the polypeptide domain of an IL27Rα-binding molecule of the present disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, the α functionality and any reactive side chains can be protected with acid-labile or base-labile groups that are stable under conditions that link amide bonds but can be easily cleaved without damaging the resulting peptide chain.

[0146] In solid-phase synthesis, either the N-terminal or C-terminal amino acid can be attached to a suitable support material. Suitable support materials are those that are inert to the reagents and reaction conditions for the stepwise condensation and cleavage reactions of the synthesis process and are insoluble 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, typically performed using an automated peptide synthesizer, can be performed according to conventional methods to sequentially attach protected amino acids.

[0147] At the end of solid-phase synthesis, the peptide is cleaved from the support material with simultaneous cleavage of the side chain protecting groups. The resulting peptide can be purified by a variety of chromatographic methods, including, but not limited to, hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high pressure liquid chromatography (HPLC), and reverse-phase HPLC.

[0148] Recombinant production Alternatively, the polypeptide domain of the IL27Rα-binding molecule of the present disclosure may be produced by recombinant DNA technology. In a typical implementation of recombinant production of a polypeptide, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector appropriate for the host cell in which expression will occur, and the nucleic acid sequence is operably linked to one or more expression control sequences encoded by the vector and functional in the target host cell. The recombinant protein may be recovered by disrupting the host cells or, if a secretory leader sequence (signal peptide) is incorporated into the polypeptide, from the cell culture medium. The recombinant protein may be purified and concentrated for further use, including incorporation.

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

[0150] A nucleic acid molecule is not limited to a sequence encoding a polypeptide. It may also include some or all of the non-coding sequences upstream or downstream from the coding sequence (e.g., the coding sequence of the polypeptide domain of an IL27Rα-binding molecule). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. For example, nucleic acid molecules can be prepared by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0151] Nucleic acid molecules encoding polypeptide domains of IL27Rα-binding molecules (and fusions thereof) may comprise naturally occurring sequences or may comprise sequences that differ from those occurring in nature but that, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules may be composed 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 found within these types of nucleic acids. Furthermore, nucleic acid molecules may be double-stranded or single-stranded (i.e., either the sense strand or the antisense strand).

[0152] Nucleic acid sequences encoding polypeptide domains of IL27Ra-binding molecules may be obtained from various commercial suppliers that offer custom synthesis of nucleic acid sequences. Amino acid sequence variants of the human IL27Ra-binding molecules of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code, as known in the art. Such variants are insertions, substitutions, and / or designated deletions of residues as mentioned. Any combination of insertions, substitutions, and / or designated deletions can be made to arrive at the final construct, provided that the final construct possesses the desired biological activity as defined herein.

[0153] Methods for constructing DNA sequences encoding polypeptide domains of IL27Rα-binding molecules and expressing these sequences in an appropriately 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 domains of IL27Rα-binding molecules can also be made using standard recombinant techniques. For deletions or additions, the nucleic acid molecule encoding the polypeptide domain of an IL27Rα-binding molecule is optionally digested with an appropriate restriction endonuclease. The resulting fragments can be expressed directly or further manipulated, for example, by ligating to a second fragment. Ligation may be facilitated if the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, although blunt-ended fragments can also be ligated. Nucleic acids generated by PCR can also be used to generate a variety of mutant sequences.

[0154] The polypeptide domains of the IL27Rα-binding molecules of the present disclosure can be produced directly recombinantly or as fusion polypeptides with heterologous polypeptides, such as signal sequences or other polypeptides having specific cleavage sites at the N- or C-terminus of the mature IL27Rα-binding molecule. Generally, the signal sequence may be a component of the vector or part of the coding sequence inserted into the vector. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In some embodiments, the signal sequence is the signal sequence naturally associated with the IL27Rα-binding molecule (i.e., the human IL27Rα signal sequence). The inclusion of a signal sequence depends on whether it is desired to secrete the IL27Rα-binding molecule from the recombinant cell in which it is produced. If the cell of choice is prokaryotic, it is generally preferred that the DNA sequence does not encode a signal sequence. If the cell of choice is eukaryotic, it is generally preferred that a signal sequence is encoded, most preferably a wild-type IL-2 signal sequence. Alternatively, signal sequences from secreted polypeptides of the same or related species may be suitable, as well as heterologous mammalian signal sequences, such as viral secretory leaders, e.g., the herpes simplex gD signal. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, the α-mating factor secretory signal sequence may be used for extracellular secretion of the IL27Rα-binding molecule into the culture medium, as described by Singh, U.S. Patent No. 7,198,919 B1.

[0155] When the polypeptide domain of an IL27Rα-binding molecule to be expressed is expressed as a chimera (e.g., a fusion protein comprising an IL27Rα-binding molecule and a heterologous polypeptide sequence), the chimeric protein may be encoded by a hybrid nucleic acid molecule comprising a first sequence encoding all or a portion of the polypeptide domain of the IL27Rα-binding molecule and a second sequence encoding all or a portion of the heterologous polypeptide. For example, the polypeptide domain of an IL27Rα-binding molecule described herein may contain a hexa-histidine tag to facilitate purification of bacterially expressed proteins. (SEQ ID NO:195) and hexa-histidine to facilitate purification of the protein expressed in eukaryotic cells. (SEQ ID NO:195) , hemagglutinin, or Fc tag. The first and second should not be construed as limitations on the orientation of the elements of the fusion protein; the heterologous polypeptide can be linked to either the N-terminus and / or C-terminus of the polypeptide domain of the IL27Ra-binding molecule. For example, the N-terminus may be linked to a targeting domain, and the C-terminus to a hexahistidine tag. (SEQ ID NO:195) It may be connected to a purification handle.

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

[0157] In some embodiments, nucleic acid sequences encoding polypeptide domains of IL27Ra-binding molecules may be "codon-optimized" to facilitate expression in particular host cell types. Techniques for codon optimization in a wide variety of expression systems, including mammalian, yeast, and bacterial host cells, are well known in the art, and online tools exist to provide codon-optimized sequences for expression in various host cell types. See, for example, Hawash, et al., (2017) 9:46-53, and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols , edited by David Hacker (Human Press, New York). Additionally, there are a variety of web-based online software packages freely available to aid in the preparation of codon-optimized nucleic acid sequences.

[0158] Expression vector Once assembled (by synthesis, site-directed mutagenesis, or another method), the nucleic acid sequence encoding the polypeptide domain of the IL27Rα-binding molecule is inserted into an expression vector. A variety of expression vectors are available for use in various host cells and are typically based on the host cell for expression. Expression vectors typically include, but are 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, integrating vectors, and the like. A plasmid is an example of a non-viral vector. To facilitate efficient expression of the recombinant polypeptide, the nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory control sequences that function in the selected expression host.

[0159] Expression vectors typically contain a selection gene, also called a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing a 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 complement auxotrophic deficiencies, or (c) proteins that supply important nutrients unavailable from complex media.

[0160] Expression vectors for the polypeptide domains of the IL27Ra-binding molecules of the present disclosure contain regulatory sequences that are recognized by the host organism and are operably linked to the nucleic acid sequence encoding the polypeptide domain of the IL27Ra-binding molecule. The terms "regulatory control sequence," "regulatory sequence," or "expression control sequence" are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego CA USA). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. In selecting an expression control sequence, a variety of factors understood by those skilled in the art should be considered. These include, for example, the relative strength of the sequence, its controllability, and, particularly with respect to potential secondary structures, compatibility with the actual DNA sequence encoding the present IL27Rα-binding molecule.

[0161] In some embodiments, the regulatory sequence is a promoter, and the promoter is selected based, for example, on the cell type in which expression is desired. A promoter is an untranslated sequence located upstream (5') of the start codon of a structural gene (generally within about 100-1000 bp) that controls the transcription and translation of a specific operably linked nucleic acid sequence. Such promoters are typically divided into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters 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 a nutrient or a change in temperature. Numerous promoters recognized by a variety of potential host cells are known.

[0162] The T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoter can be used in mammalian cells.Similarly, in the case of higher eukaryotes, tissue-specific promoters and cell type-specific promoters are widely available.These promoters are named after their ability to induce the expression of nucleic acid molecules in certain tissues or cell types in the body.Those skilled in the art are familiar with a great number of promoters and other regulatory elements that can be used to induce nucleic acid expression.

[0163] Transcription from vectors in mammalian host cells may be controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., murine stem cell virus), hepatitis B virus, and most preferably simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoters, heat shock promoters, provided that such promoters are compatible with the host cell system. Conveniently, the early and late promoters of the SV40 virus are obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0164] Transcription in higher eukaryotes is often increased by inserting enhancer sequences into vectors. Enhancers are cis-acting DNA elements, usually about 10 to 300 bp, that act on promoters to increase transcription. Enhancers are relatively orientation- and position-independent, being found 5' and 3' to transcription units, within introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers derived from eukaryotic viruses are typically used. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers may be spliced ​​into expression vectors 5' or 3' to the coding sequence, 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, or sometimes the 3' untranslated region, of eukaryotic or viral DNA or cDNA.Standard techniques are used to construct suitable vectors containing one or more of the above-listed components.

[0165] In addition to sequences facilitating transcription of the inserted nucleic acid molecule, vectors may contain an origin of replication and other genes encoding selectable markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells in which it is expressed, thus allowing 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 ascertain whether a particular regulatory element or selectable marker is suitable for use in a particular experimental situation. Correct assembly of an expression vector can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in an appropriate host.

[0166] host cell Additionally, the present disclosure provides prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding a polypeptide domain of an IL27Ra-binding molecule. The cells of the present disclosure are transfected cells, i.e., cells into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding a polypeptide domain of an IL27Ra-binding molecule, has been introduced by recombinant DNA techniques. Progeny of such cells are also considered within the scope of the present disclosure.

[0167] Host cells are typically selected based on compatibility with the selected expression vector, toxicity of the product encoded by the DNA sequence of the IL27Rα-binding molecule, secretion characteristics, ability to properly fold the polypeptide, fermentation or culture requirements, and ease of purification of the product encoded by the DNA sequence. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotes.

[0168] In some embodiments, recombinant polypeptide domains of IL27Rα binding molecules or biologically active variants thereof can also be produced in eukaryotic organisms, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors available 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 the 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), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

[0169] Examples of useful mammalian host cell lines include mouse L cells (LM[TK-], ATCC #CRL-2648), SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651); human embryonic 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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and human hepatoma line (HepG2). In mammalian cells, control functions of expression vectors are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.

[0170] The polypeptide domain of the IL27Rα-binding molecule may be produced in a prokaryotic host, such as the bacterium Escherichia coli, or in a eukaryotic host, such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). Those skilled in the art can make such decisions. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0171] In some embodiments, the recombinant polypeptide domain of an IL27Rα-binding molecule may be glycosylated or non-glycosylated, depending on the host organism used to produce the IL27Rα-binding molecule. When bacteria are selected as the host, the polypeptide domain of the IL27Rα-binding sdAb may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser (NXS) or Asn-X-Thr (NXT), where X is any amino acid except proline. In such cases, it may be desirable to eliminate the N-linked glycosylation motif by modifying the sequence of such an N-linked glycosylation motif to prevent glycosylation. In some embodiments, elimination of the Asn-X-Ser (NXS) N-linked glycosylation motif can be achieved by incorporating conservative amino acid substitutions of the Asn(N) and / or Ser(S) residues of the Asn-X-Ser (NXS) N-linked glycosylation motif. In some embodiments, elimination of the Asn-X-Thr(NXT) N-linked glycosylation motif can be achieved by incorporating conservative 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 a prokaryotic expression system is used to produce a recombinant Criss recombinant IL27Rα-binding sdAb, sequence modifications to eliminate N-linked glycosylation sites may not be necessary because the prokaryotic host cell does not provide the machinery for glycosylation of recombinant proteins.

[0172] For additional expression systems, both prokaryotic and eukaryotic, 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 Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0173] Transfection The expression construct can be introduced into host cells to produce the recombinant polypeptide domain of the IL27Ra-binding molecule disclosed herein, or to produce a biologically active mutein thereof. The vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection methods. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0174] To facilitate the transfection of target cells, target cells can be directly exposed to non-viral vectors under conditions that facilitate the uptake of non-viral vectors. Examples of conditions that facilitate the uptake of exogenous nucleic acid 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, and magnetic field (electroporation).

[0175] cell culture Cells may be cultured in conventional nutrient media, modified as appropriate for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence. Mammalian host cells can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), minimal essential medium (MEM), Sigma, RPMI 1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma), are suitable for culturing host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0176] Recombinant protein recovery If a secretory leader sequence is used, recombinantly produced IL27Rα-binding polypeptides can be recovered from the culture medium as secreted polypeptides. Alternatively, IL27Rα-binding polypeptides can also be recovered from host cell lysates. To inhibit proteolysis during purification, protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) may be used during recovery from cell lysates, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0177] purification Various purification processes, such as affinity chromatography, are known and utilized in the art. Affinity chromatography typically utilizes highly specific binding sites present on biological macromolecules to separate molecules capable of binding to a specific ligand. The ligand is covalently attached to an insoluble porous support medium in such a way that the ligand is specifically presented to a protein sample, thereby utilizing the natural specific binding of one molecular species to separate and purify a second species from the mixture. Antibodies are commonly used in affinity chromatography. A size selection process may also be employed, for example, gel filtration chromatography (also known as size exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to size. 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 determine the size of proteins that can be separated by the column.

[0178] Recombinant polypeptide domains of IL27Rα-binding molecules produced by transformed hosts can be purified according to any suitable method: IL27Rα-binding molecules may be isolated from inclusion bodies formed in E. coli using cation exchange, gel filtration, and / or reverse-phase liquid chromatography, or from conditioned medium derived from either mammalian or yeast cultures producing a particular IL27Rα-binding molecule.

[0179] The recombinant polypeptide in a substantially purified form can be used, for example, as a therapeutic agent, as described herein.

[0180] The biological activity of the recombinant polypeptide domain of the IL27Rα-binding molecule produced as described above can be determined by competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plots, non-linear curve fitting programs, and competitive binding assays); 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 by commercially available instrumentation from GE Healthcare Bio-Sciences, e.g., Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA). 01752); solution-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays).

[0181] How to use Inhibition of IL27Rα activity In one aspect, the disclosure provides a method of modulating the activity of IL27Rα-expressing cells by administering to a subject an IL27Rα-binding molecule in an amount sufficient to interfere with the activity of a receptor that includes IL27Rα. Additionally, the disclosure provides a method of modulating the activity of IL27Rα-expressing cells in a mixed cell population, comprising contacting the cell population in vivo and / or ex vivo with an IL27Rα-binding molecule or complex of the disclosure in an amount sufficient to interfere with the activity of a receptor that includes IL27Rα.

[0182] Identifying, isolating, enriching, or depleting IL27Rα+ cells In one aspect, the present disclosure provides methods for using the disclosed IL27Rα-binding molecules in processes for isolating, enriching, or depleting IL27Rα+ cells from a biological sample containing IL27Rα+ cells. The biological sample may include blood-derived cells, e.g., PBMCs, T cells, B cells, of cell culture origin, or of tissue origin, such as brain or bone marrow. Suitable processes for isolating, enriching, or depleting IL27Rα+ cells include centrifugation, filtration, magnetic cell sorting, and fluorescent cell sorting by techniques well known in the art. Furthermore, the present disclosure provides methods for treating a subject suffering from a disease, disorder, or condition by administering a therapeutically effective amount of a cell product enriched or depleted in IL27Rα+ cells using an IL27Rα-binding molecule as described herein.

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

[0184] In another embodiment, the selection procedure uses IL27Rα-binding molecules of the present disclosure (e.g., IL27Rα-binding VHHs) conjugated to magnetic particles, which result in magnetic labeling of IL27Rα+ cells for use in a magnetic cell separation procedure. In one embodiment, the method includes the steps of: (a) conjugating one or more IL27Rα-binding molecules of the present disclosure (e.g., IL27Rα-binding VHHs) to magnetic particles; (b) contacting a biological sample with a quantity of magnetic particles conjugated to IL27Rα-binding molecules to create a mixture; (c) subjecting the mixture to a magnetic field such that the magnetically labeled IL27Rα+ cells are retained; (d) removing non-magnetically labeled cells from the mixture; and (e) removing the magnetic field to isolate the IL27Rα+ cells.

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

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

[0187] kit The present disclosure also contemplates kits comprising pharmaceutical compositions of IL27Rα-binding molecules. The kits generally take the form of physical structures housing various components, as described below, and can be utilized, for example, in practicing the methods described above. The kits of the present disclosure can be designed for conditions (e.g., refrigeration or freezing) necessary to properly maintain the components housed in the kit. The kits may further comprise a label or package insert identifying the components in the kit and instructions for use. Each component of the kit may be enclosed in an individual container, or the various containers may all be in a single package. The label or package insert may include manufacturer information, such as a lot number and expiration date. The label or package insert may, for example, be integrated into the physical structure housing the component, be separately contained within the physical structure, or be affixed to a component of the kit (e.g., an ampoule, syringe, or vial). The label or package insert may be provided in a physical form or on a computer-readable medium. In some embodiments, the actual instructions are not present in the kit, but the kit provides means for obtaining the instructions from a remote source, e.g., via an internet site, including obtaining the instructions by secure access through provision of a password (or a scannable code, such as a barcode or QR code, on the container of the IL27Rα-binding molecule or on the surface of the containing kit), in accordance with government regulations (e.g., HIPAA). [Example]

[0188] The following examples are presented so as to fully disclose and describe to those of skill in the art how to make and use the present IL27Rα-binding molecules and are not intended to limit the scope of what the inventors regard as IL27Rα-binding molecules, nor are they intended to represent that the experiments below have been performed or are all that could be performed. It should be understood that an exemplary description written in the present tense may not necessarily be performed, but that the description could be performed to obtain the data, etc., described therein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should account for. It is expected that variations of the specifically described procedures used will be apparent to those of skill in the art, who will be able to employ such variations as appropriate. Accordingly, the IL27Rα-binding molecules may be performed otherwise than as specifically described herein, and the present invention is intended to include all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law.

[0189] Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. 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; mM = millimolar; M = molar; kDa = kilodalton; im = intramuscular (into the muscle); ip = intraperitoneal (into the peritoneal cavity); SC or SQ = subcutaneous (under the skin); QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; 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's medium; GC = genome copies; EDTA = ethylenediaminetetraacetic acid; PBMC = primary peripheral blood mononuclear cells; FBS = fetal bovine serum; FCS = fetal calf serum; HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LPS = lipopolysaccharide; ATCC = American Type Culture Collection.

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

[0191] Synthetic DNA sequences encoding the antigen were inserted into the pFUSE_hIgG1_Fc2 vector (Generay Biotechnology) and transfected into HEK293F mammalian host cells for expression. The antigen was expressed as an Fc fusion protein, which was purified using protein A chromatography. The antigen was diluted in 1x PBS (approximately 1 mg total antigen). Quality was assessed by SDS-PAGE to ensure sufficient purity (>80%) for immunization. Camels were acclimated in the facility for at least 7 days prior to immunization. Immunization with the antigen was performed using weekly antigen administrations 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 1x PBS was slowly added to the mortar while grinding with a pestle. The sample was ground until the antigen emulsified, became milky white, and was difficult to disperse. For six subsequent immunizations (weeks 2–7) in the immunization protocol, immunogens were prepared as described above, except that incomplete Freund's adjuvant (IFA) was used instead of CFA. Camels were injected subcutaneously with approximately 2 ml of emulsified antigen at at least six sites, for a total of approximately 10 ml per camel. When the antigen was injected, the needle was maintained in the subcutaneous space for approximately 10–15 seconds after each injection to avoid leakage of the emulsion.

[0192] Example 2. Phage library construction Three days after the final injection in the immunization protocol, blood samples were collected from the camels. RNA was extracted from the blood and transcribed into cDNA. A ∼900 bp reverse-transcribed sequence encoding the VH-CH1-hinge-CH2-CH3 construct was isolated from the desired ∼700 bp fragment encoding the VHH-hinge-CH2-CH3 species. The purified ∼700 bp fragment was amplified by nested PCR. The amplified sequence was digested with Pst1 and Not1. The ∼400 bp PST1 / Not1-digested fragment was inserted into a Pst1 / Not1-digested pMECS phagemid vector so that the VHH coding sequence was in-frame with the DNA sequence encoding the HA / His sequence. The PCR-generated sequence and the pMECS phagemid vector were digested with PstI and NotI and then ligated into pMECS / Nb recombinant. After ligation, the product was transformed into E. coli TG1 cells by electroporation. Transformants were concentrated in growth medium and then transferred to 2YT+2% glucose agar plates.

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

[0194] Example 4: Identification of antibodies that exhibit specific binding to IFNgR1: After completing the biopanning in Example 3, three 96-well plates of individual phage clones were isolated to perform periplasmic extract ELISA (PE-ELISA) on IL27Rα-coated plates to identify positive VHH binders that selectively bound to IFNgR1. The 96-well plates were coated with IL27Rα and PBS under the same conditions. The wells were then blocked for 1 hour at 37°C. Then, 100 μl of the 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 for 1 hour at 37°C. The plates were developed with TMB substrate. The reaction was stopped by adding H2SO4. The absorbance at 450 nm was read using a microtiter plate reader. Antibodies that showed an absorbance of the antigen-coated wells that was at least three times higher than that of the PBS-coated control were defined as exhibiting specific binding to IL27Rα. Positive clones were sequenced and sequences analyzed to identify unique clonotypes.

[0195] Example 5. Assessment of binding affinity via surface plasmon resonance Representative examples from each hIL27Rα VHH clonotype generated according to Examples 1-3 were selected for binding assessment via SPR as follows. Assessment of the binding affinity of hIL27Rα binding molecules corresponding to SEQ ID NOs. 2-27 was performed using surface plasmon resonance (SPR) generally according to the following procedure. All experiments were performed on a Biacore T200 instrument equipped with a Protein A-derivatized sensor chip (Cytiva) in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20), and 3 mM EDTA (HBS-EP+ buffer). Mono-Fc VHH ligands were flowed at 5 μl / min for variable times ranging from 18 to 300 seconds to reach the capture loads listed in the table below. After ligand capture, a two-fold dilution series of the extracellular domain of the IL27Rα receptor modified to incorporate a C-terminal poly-His sequence, typically containing at least five concentrations ranging from 1 μM to 1 nM, was injected in high-performance or single-cycle kinetics mode. Surface regeneration was achieved by injecting 10 mM glycine-HCl, pH 1.5 (60 s, 50 μL / min). Buffer-subtracted sensograms were processed using Biacore T200 Evaluation Software to calculate rate and affinity constants (k a , k d , K. D ) was globally fitted using a 1:1 Langmuir binding model (bulk shift set to 0). MAX <100 RU indicates a surface density compatible with kinetic analysis. max Calculations were generated using the formula: Rmax = load (RU) x valency of ligand x (molecular weight of analyte / molecular weight of ligand). Surface activity was defined as the ratio of experimental Rmax / calculated. The results of these binding affinity experiments are shown in Table 6.

[0196] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations will be suggested to those skilled in the art in light of the examples and embodiments described herein, and are within the spirit and scope of this application and the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Claims

1. 1. An interleukin 27 receptor alpha (IL27Rα) binding molecule that specifically binds to the extracellular domain of IL27Rα, comprising: a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:50, a CDR2 comprising the amino acid sequence of SEQ ID NO:51, and a CDR3 comprising the amino acid sequence of SEQ ID NO:52; or CDR1 comprising the amino acid sequence of SEQ ID NO:26, CDR2 comprising the amino acid sequence of SEQ ID NO:27, and CDR3 comprising the amino acid sequence of SEQ ID NO:28; or CDR1 comprising the amino acid sequence of SEQ ID NO:68, CDR2 comprising the amino acid sequence of SEQ ID NO:69, and CDR3 comprising the amino acid sequence of SEQ ID NO:70; or CDR1 comprising the amino acid sequence of SEQ ID NO:80, CDR2 comprising the amino acid sequence of SEQ ID NO:81, and CDR3 comprising the amino acid sequence of SEQ ID NO:82; or CDR1 comprising the amino acid sequence of SEQ ID NO:86, CDR2 comprising the amino acid sequence of SEQ ID NO:87, and CDR3 comprising the amino acid sequence of SEQ ID NO:

88. IL27Rα binding molecule comprising a single domain antibody comprising:

2. V H The IL27Rα-binding molecule of claim 1, wherein said IL27Rα-binding molecule is H.

3. The IL27Rα binding molecule of claim 1, wherein the single domain antibody is humanized or comprises CDRs grafted onto a heterologous framework.

4. The IL27Rα binding molecule of claim 1, further comprising a labeling agent, an imaging agent, and / or a therapeutic agent.

5. A kit comprising the IL27Rα binding molecule of claim 1.

6. The IL27Rα binding molecule of claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:50, CDR2 comprises the amino acid sequence of SEQ ID NO:51, and CDR3 comprises the amino acid sequence of SEQ ID NO:

52.

7. The IL27Rα binding molecule of claim 1, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:

10.

8. The IL27Rα binding molecule of claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:26, CDR2 comprises the amino acid sequence of SEQ ID NO:27, and CDR3 comprises the amino acid sequence of SEQ ID NO:

28.

9. The IL27Rα binding molecule of claim 8, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:

2.

10. The IL27Rα binding molecule of claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:68, CDR2 comprises the amino acid sequence of SEQ ID NO:69, and CDR3 comprises the amino acid sequence of SEQ ID NO:

70.

11. The IL27Rα binding molecule of claim 10, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:

16.

12. The IL27Rα binding molecule of claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:80, CDR2 comprises the amino acid sequence of SEQ ID NO:81, and CDR3 comprises the amino acid sequence of SEQ ID NO:

82.

13. The IL27Rα binding molecule of claim 12, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:

20.

14. The IL27Rα binding molecule of claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO:86, CDR2 comprises the amino acid sequence of SEQ ID NO:87, and CDR3 comprises the amino acid sequence of SEQ ID NO:

88.

15. The IL27Rα binding molecule of claim 14, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO:22.

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