CN116723859A - IL27Rα binding molecules and methods of use - Google Patents

IL27Rα binding molecules and methods of use Download PDF

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Publication number
CN116723859A
CN116723859A CN202180068078.5A CN202180068078A CN116723859A CN 116723859 A CN116723859 A CN 116723859A CN 202180068078 A CN202180068078 A CN 202180068078A CN 116723859 A CN116723859 A CN 116723859A
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binding
cells
cell
polypeptide
binding molecule
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R·卡斯特莱茵
D·罗卡姆
P·J·卢帕杜斯
S·维欧娜
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Sindkain Co ltd
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Sindkain Co ltd
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Priority claimed from PCT/US2021/044576 external-priority patent/WO2022031870A1/en
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Abstract

The present application relates to bioactive molecules including single domain antibodies (sdabs) that specifically bind to the extracellular domain of human IL27 ra, and compositions, including such antibodies, and methods of use thereof.

Description

IL27Rα binding molecules and methods of use
Cross Reference to Related Applications
The disclosure of which is incorporated herein by reference in its entirety for all purposes, claims priority from U.S. provisional application No. 63/061,562 filed 8/5/2020, U.S. provisional application No. 63/078,745 filed 9/15/2020, and U.S. provisional application No. 63/135,884 filed 1/11/2021.
Technical Field
The present application relates to bioactive molecules, including single domain antibodies that specifically bind to the extracellular domain of IL27 receptor alpha (IL 27 ra), compositions, and methods of use thereof, including such single domain antibodies.
Background
Cytokine IL-27 is a heterodimeric cytokine consisting of two non-covalently linked subunits, p28 and EBI 3. The p28 subunit belongs to the 4-helix bundle cytokine family, while EBI3 is the shortest possible form of the soluble cytokine receptor, with two typical cytokine binding domains (Pflnz S, et al, immunity.2002, month 6; 16 (6): 779-90).
The interleukin-27 receptor (IL 27R) is a type I cytokine receptor for interleukin-27 (IL 27). IL27R is a heterodimer, consisting of an IL27R alpha subunit and glycoprotein 130 (IL 6 Rb). IL27 is expressed by antigen presenting cells and induces T cell differentiation in different populations of the immune system. Binding of IL27 to IL27R initiates intracellular signaling of several Jak family kinases that induce phosphorylation of STAT1 and STAT 3. In activated T cells, IL27 signals predominantly through STAT3[23], whereas in memory B cells it signals predominantly through STAT 3. IL27 has been shown to have pro-inflammatory and anti-inflammatory properties, with pro-inflammatory or anti-inflammatory responses being affected by the environment of the cells expressing IL 27R.
The IL27R alpha subunit (also known as the TCCR-or WSX-1 receptor) is a proprietary subunit of the IL27 receptor. Mature (signal peptide removed) IL27 ra is a 604 amino acid polypeptide with an extracellular domain of 484 amino acids. The extracellular domain of IL27rα has 5 domains: D1-D5. D1 and D2 are the major cytokine binding domains, while the type III fibronectin (Fn 3) domains D3, D4 and D5 are involved in ligand recognition to a lesser extent. Based on structural analysis, fn3 domains do not contribute to binding in the complex when IL27 ligand binds. Although domains D1 and D2 are highly conserved, the sequence of the Fn3 domain is more variable.
IL27 exhibits a high (nanomolar) affinity for the IL27Ra subunit. The [ IL27/IL27Rα ] complex associates with IL27Ra to complete the IL27 receptor signaling complex. gp130 binds to the [ IL27/IL27Rα ] complex much weaker than the interaction between IL-27 and IL-27R. (Pflnz S, et al, J Immunol.2004, 15, 2, 172 (4): 2225-31), which is somewhat classical in sharing cytokine receptor subunits. Due to the 'C' shape of each receptor, the D5 domain of IL-27R and the D6 domain of gp130 are in close proximity at the membrane. This is required for the receptor complex to be able to trigger JAK binding at the intracellular domains of both receptors.
Although monoclonal antibodies are the most widespread reagents for detecting and quantifying proteins, monoclonal antibodies are large molecules of about 150kDa, the size of which can potentially limit their use in assays that compete with multiple reagents for near epitope recognition. A unique class of immunoglobulins contains heavy chain domains but lacks light chain domains (commonly referred to as heavy chain "antibodies (HCAbs)) present in camelids, including dromedaries, bactrian camels, wild dromedaries, llamas, alpacas, camelsAnd alpaca, and cartilaginous fish such as shark. The isolated variable domain region of an HCAb is called VHH (an abbreviation reflecting its "variable-heavy" structure) or +. >(Ablynx). Single domain VHH antibodies have the advantage of being small in size (-12-14 kD) and having a molecular weight of about one tenth that of conventional mammalian IgG class antibodies, which facilitates binding of these VHH molecules to the antigenic determinants of the target, which may not be possible with conventional monoclonal IgG formatsNear these determinants (Ingram et al, 2018). Furthermore, VHH single domain antibodies typically have high thermal stability characteristics that help distribute agents to geographical areas where it is difficult or impossible to maintain the cold chain. These properties, particularly binding, do not require a heavy/light chain pairing (as in the case of IgG antibodies) and simple phage display discovery methods for simple manufacture (as in bacterial expression systems), make VHH single domain antibodies useful in a variety of applications, including the development of imaging agents and therapeutic agents.
Disclosure of Invention
The present invention provides polypeptides that specifically bind to IL27 Ra.
The present invention provides polypeptides that specifically bind to the extracellular domain of IL27 Ra.
The present invention provides an IL27Ra binding molecule that specifically binds to the extracellular domain of human IL27Ra (hIL 27 Ra).
In some embodiments, the IL27Ra binding molecule comprises a single domain antibody (sdAb) that specifically binds to the extracellular domain of human IL27 Ra.
In some embodiments, the IL27Ra binding molecule is an sdAb comprising a set of CDRs corresponding to CDR1, CDR2, and CDR3, as shown in the rows of table 1 below.
In some embodiments, the IL27Ra binding molecule comprises CDR1, CDR2, and CDR3 as set forth in the rows of table 1 below, wherein 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, or have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, relative to the sequences set forth in the rows of table 1 below.
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%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity (or identical except that 1, 2, 3, or 4 amino acids are optional conservative amino acid substitutions) or 100% identity to any of the polypeptide sequences of SEQ ID NOs 2-25, as shown in table 1 below.
In some embodiments, the foregoing CDR sets are incorporated into a humanized VHH framework to provide a "humanized" sdAb IL27Ra binding molecule.
The invention also provides methods for chemical or recombinant processes for preparing the IL27Ra binding molecules of the invention.
The invention also provides nucleic acids encoding IL27Ra binding molecules. Table 2 below provides examples of DNA sequences encoding IL27Ra binding molecules as described herein.
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In some embodiments, the IL27Ra is murine IL27Ra.
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 (ml27 Ra).
In some embodiments, the IL27Ra binding molecule is an sdAb comprising a set of CDRs corresponding to CDR1, CDR2, and CDR3, as shown in the rows of table 3 below.
In some embodiments, the IL27Ra binding molecule comprises CDR1, CDR2, and CDR3 as set forth in the rows of table 3 below, wherein 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, or have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, relative to the sequences set forth in the rows of table 3 below.
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%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity (or identical except that 1, 2, 3, or 4 amino acids 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.
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In some embodiments, the foregoing CDR sets are incorporated into a humanized VHH framework to provide a "humanized" sdAb IL27ra binding molecule.
The invention also provides methods for preparing the chemical or recombinant processes of the IL27Rα binding molecules of the invention.
The invention also provides nucleic acids encoding IL27Ra binding molecules. Table 4 below provides examples of DNA sequences encoding hIL27Ra binding molecules, as set forth in Table 3 above.
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The invention further provides recombinant viral and non-viral vectors comprising nucleic acids encoding the IL27ra binding molecules of the invention or CDRs of the IL27ra binding molecules of the invention.
The invention also provides host cells comprising recombinant viral and non-viral vectors comprising nucleic acids of the IL27ra binding molecules of the invention or CDRs of the IL27ra binding molecules of the invention.
The invention also provides host cells comprising recombinant viral and non-viral vectors comprising nucleic acids of the IL27ra binding molecules of the invention or CDRs of the IL27ra binding molecules of the invention.
The invention also provides kits comprising the IL27Rα binding molecules of the invention.
In another aspect, the invention provides constructs for identifying cells expressing IL27ra, wherein the IL27ra binding molecule is coupled to one or more imaging agents, optionally through a chemical or polypeptide linker. The invention also provides a method of use as described above for identifying cells expressing IL27ra in a subject, the method comprising administering to a subject in need of treatment an effective amount of an IL27ra binding molecule coupled to an imaging agent, and assessing whether the subject has an imaging agent coupled to the IL27ra binding molecule.
In another aspect, the invention provides IL27 ra binding molecules that are modified in vivo to extend the duration of action, wherein the IL27 ra binding molecules are conjugated to one or more carrier molecules.
The present invention provides IL27rα binding molecules comprising polypeptide sequences that specifically bind to the extracellular domain of IL27rα, and methods of use thereof for isolating, depleting or enriching cells expressing IL27rα in a biological sample.
Detailed Description
Introduction to the invention
In order that the invention may be more readily understood, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be read in light of the knowledge of one skilled in the art.
Before describing the methods and compositions of the present invention, it is to be understood that this invention is not limited to the described methods or compositions, as such may, of course, vary.
Where a range of values is provided, it is also contemplated that each intervening value, in the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. The invention also includes smaller ranges between any given value or intervening value in a given range and any other given or intervening value in that given range. Any explicitly excluded limit in a stated range may be the upper and lower limits of those smaller ranges independently, and the invention also includes ranges not including any, including either or both limits. Where a range is set to include one or both of the limits, the invention also includes ranges excluding either or both of those included limits.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application, some of the potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides and equivalents thereof known to those skilled in the art, such as polypeptides, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application. Furthermore, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.
It is understood that in the present disclosure, amino acids are referred to according to single letter or three letter codes. For the convenience of the reader, single letter and three letter amino acid codes are provided in table 5 below:
standard methods of molecular biology are described in the scientific literature (see, e.g., sambrook and Russell (2001) & molecular cloning (Molecular Cloning), 3 rd edition, cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press), new York Cold spring harbor, and Ausubel et al (2001) & New coded guidelines for molecular biology (Current Protocols in Molecular Biology), volume 1-4, john Willi father (John Wiley and Sons, inc.) New York, N.Y., which describe cloning and DNA site-directed mutagenesis in bacterial cells (volume 1), cloning in mammalian cells and yeast (volume 2), glycoconjugates and protein expression (volume 3), and bioinformatics (volume 4)). The scientific literature describes methods for protein purification 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), "New protein science protocol (Current Protocols in Protein Science), volume.1-2, john Willi parent company (John Wiley and Sons, inc.), NY).
Definition of the definition
Unless otherwise indicated, the following terms are intended to have the following meanings. Other terms are defined throughout the specification.
Activation of: the term "activation" as used herein is used to refer to a receptor or receptor complex to reflect a biological effect produced directly and/or by participation in a multicomponent signaling cascade, which biological effect is a binding response to a ligand, resulting from the binding of an agonist ligand to the receptor.
Activity(s): the term "activity" as used herein is with respect to a molecule to describe the identity or biological or chemical property of the molecule (e.g., the extent to which the molecule binds to another molecule) or the physical property of a material or cell (e.g., a change in cell membrane potential) relative to a test system (e.g., assay). Examples of such biological functions include, but are not limited to, catalytic activity of biological agents, ability to stimulate intracellular signaling, gene expression, cell proliferation, ability to modulate immunological activity (e.g., inflammatory response). "Activity" is generally expressed as the level of biological activity of the test agent per unit, e.g. [ catalytic Activity ]][ mg protein ]][ immunocompetence ]][ mg protein ]]International units of activity (IU), [ STAT5 phosphorylation ]][ mg protein ] ][ proliferation ]][ mg protein ]]Plaque forming units (plaque forming units) (pfu), and the like. As used herein, the term proliferative activity refers to an activity that promotes cell proliferation and replication, which includes deregulated cell division observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis and angiogenesis.
Administration/administration: the terms "administration" and "administering" are used hereinInterchangeably used, refers to an act of contacting a subject, including contacting a cell, tissue, organ, or biological fluid in vitro, in vivo, and/or ex vivo of a subject with an agent (e.g., an IL27 ra binding molecule or an engineered cell expressing an IL27 ra binding molecule, a chemotherapeutic agent, an antibody, or a pharmaceutical formulation comprising one or more of the foregoing). Administration of the agent may be accomplished by any of a variety of art-recognized methods including, but not limited to, topical administration, intravascular injection (including intravenous or intra-arterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal, transmucosal, iontophoretic delivery (iontophoretic delivery), intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g., respiratory tract inhaler, including dry powder inhaler), intraocular injection, intraperitoneal injection, intralesional injection, intra-ovarian injection, intra-brain infusion or injection, intraventricular injection (ICVI), and the like. The term "administering" includes contacting the agent with a cell, tissue or organ, and contacting the agent with a liquid, wherein the liquid is in contact with the cell, tissue or organ.
Affinity for: the term "affinity" as used herein refers to the degree to which a first molecule (e.g., ligand) specifically binds to a second molecule (e.g., receptor) to balance the dissociation constant (K D ) Measured as the dissociation rate constant (k off ) And association rate constant (k) between the molecule and its target on ) Is a ratio of (2).
Agonists: in some cases, the agonist is an activator of a receptor protein that can modulate cell activation, enhance activation, sensitize a cell to activation of a second agent, or up-regulate one or more genes, proteins, ligands, receptors, biological pathways that may lead to cell proliferation or to cell cycle arrest, or expression of pathways that lead to cell death (e.g., by apoptosis)A reagent that changes receptor state, resulting in a biological response, which mimics the effect of the endogenous ligand of the receptor. The term "agonist" includes partial agonists, full agonists and superagonists. An agonist may be described as a "full agonist" when it results in a substantially complete biological response (i.e., a response associated with a naturally occurring ligand/receptor binding interaction) induced by the receptor under study, or an agonist may be described as a partial agonist. A "super-agonist" is an agonist that is capable of producing a maximum response that is greater than the endogenous agonist of the target receptor, and thus has an activity that exceeds 100% of the activity of the natural ligand. Superagonists are generally synthetic molecules that exhibit a response of greater than 110%, or greater than 120%, or greater than 130%, or greater than 140%, or greater than 150%, or greater than 160%, or greater than 170% in an evaluable quantitative or qualitative parameter of the naturally-occurring form of the molecule when evaluated at similar concentrations in a comparative assay. It should be noted that the biological effects associated with full agonists may differ in extent and/or in kind from those of partial or super-agonists. In contrast to agonists, antagonists may bind specifically to a receptor, but do not result in a signaling cascade that is normally initiated by the receptor, and may alter the effect of the agonist on the receptor. Inverse agonists refer to agents that produce a pharmacological response in the opposite direction to the agonist.
Antagonists: as used herein, the term "antagonist" or "inhibitor" refers to a molecule that acts against one or more agonists. Antagonists may prevent, reduce, inhibit or neutralize the activity of an agonist, and antagonists may also prevent, inhibit or reduce constitutive activity of a target (e.g., target receptor), even without an identified agonist. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize, or down regulate, e.g., genes, proteins, ligands, receptors, biological pathways (including immune checkpoint pathways), or cells.
Antibodies to: as used herein, the term "antibody" refers collectively to: (a) Glycosylated or non-glycosylated immunoglobulins which specifically bind to target moleculesAnd (b) immunoglobulin derivatives thereof, including but not limited to antibody fragments, such as single domain antibodies. In some embodiments the immunoglobulin derivative competes with the immunoglobulin from which it is derived for binding to the target molecule. The term antibody is not limited to antibodies from immunoglobulins, cartilage fish (including but not limited to shark) of any particular species (including murine, human, equine, camel). The term "antibody" includes antibodies that can be isolated from natural sources or from animals after immunization with antigen, as well as engineered antibodies, including monoclonal antibodies, bispecific antibodies, trispecific, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered (e.g., T cell epitope-depleted) antibodies, camelized (in the case of VHH), or molecules comprising the binding domains (e.g., CDRs) of antibodies in a non-immunoglobulin scaffold. The term "antibody" should be construed as not being limited to any particular manner of synthesis, including naturally occurring antibodies that can be isolated from natural sources, as well as engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals transformed with human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with nucleic acid constructs that result in expression of the antibodies, antibodies isolated from combinatorial antibody libraries (including phage display libraries). In one embodiment, the "antibody" is a mammalian immunoglobulin of the class IgG1, igG2, igG3 or IgG 4. In some embodiments, the antibody is a "full length antibody," which includes variable and constant domains that provide binding and effector functions. The term "single domain antibody" (sdAb) as used herein refers to an antibody fragment consisting of a monomeric variable antibody domain that is capable of specifically binding to an antigen and competing for binding with the parent antibody from which it is 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 camelidae (including camels, llamas and alpacas) (see, e.g., hamers-masterman, et al (1993) Nature 363:446-448). VHH can also be referred to as heavy chain antibody or The single domain antibodies may also be derived from non-mammalian sources, such as VHH obtained from the immunization of cartilage fish (including but not limited to shark) with IgNAR antibodies.
Biological sample: as used herein, the term "biological sample" or "sample" refers to a sample obtained (or derived) from a subject. For example, the biological sample comprises a material selected from the group consisting of: body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluid (e.g., vitreous fluid, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including enriched or cell type-specific enriched portions of immunoglobulins from one or more of these tissues.
IL27Rα cells: the terms "IL27 ra cell," "cell expressing IL27 ra," "IL27 ra positive cell," and "IL27 ra+" cell are used interchangeably herein to refer to a cell that expresses and displays an IL27 ra antigen on the extracellular surface of a cell membrane. Similarly, the terms "IL27 ra negative cells", "IL27 ra-cells" are used interchangeably herein to describe cells on the cell membrane that do not express or display an IL27 ra antigen.
CDRThe term "CDR" or "complementarity determining region" as used herein refers to a discontinuous antigen binding site found within the variable regions of heavy and light chain immunoglobulin polypeptides. CDRs have been described by Kabat et al, J.biol. Chem.252:6609-6616 (1977); kabat et al, (1991) U.S. department of health and public service, entitled "immunology hot protein sequence (Sequences of Proteins ofImmunological Interest)" (also referred to herein as "Kabat 1991" or "Kabat"); from Chothia et al, J.mol. Biol.196:901-917 (also referred to herein as "Chothia"); and MacCallum et al, (1996) J.mol.biol.262:732-745, wherein the definition includes overlapping or subsets of amino acid residues when compared to each other. However, the definition of CDRs referring to an antibody or grafted antibody or variant thereof is intended to fall within the scope of the definition or terms used herein. In the context of the present invention, unless otherwise indicated, a CDRNumbering of the positions is provided according to Kabat or a hybrid of Kabat and Chothia numbering.
Equivalent to: as used herein, the term "comparable" is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, a first measurement of an evaluable quantitative parameter and a second measurement of the evaluable parameter are considered "comparable" when they do not deviate from a range where a person skilled in the art would consider that no statistically significant difference between the two results would occur. In some cases, a measurement may be considered "comparable" if it deviates from another by less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 10%, or less than 7%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%. In certain embodiments, a measurement is comparable to a reference standard if the measurement deviates from the reference standard by less than 15%, or by less than 10%, or by less than 5%.
Conservative amino acid substitutions: as used herein, the term "conservative amino acid substitution" refers to an amino acid substitution that changes a given amino acid to a different amino acid having similar biochemical characteristics (e.g., charge, hydrophobicity, and size). For example, amino acids in the following groups may be considered as conserved amino acids with each other: (1) hydrophobic amino acid: 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 acid: lysine and arginine; and (4) an acidic amino acid: aspartic acid and glutamic acid.
Derived from: the term "derived from" as used herein in the context of amino acid sequences is intended to mean that the polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid and is not intended to be limited to the source or method of preparing the protein or nucleic acid. For example, the term "derived from" includesA homologue or variant of a reference amino acid or DNA sequence.
Effective Concentration (EC) as used herein, the term "effective concentration" or its abbreviation "EC" is used interchangeably to mean the concentration of an agent in an amount sufficient to produce a change in a given parameter in a test system. The abbreviation "E" refers to the magnitude of a given 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 reaction is expressed as a factor of the concentration of the test agent ("C"). In the context of biological systems, the term E Maximum value Refers to the maximum magnitude of a given biological effect observed in a saturated concentration in response to activating a test agent. When a subscripted abbreviation EC (e.g., EC 40 、EC 50 Etc.), subscript refers to E at which a biological response is observed Maximum value Is a percentage of (c). For example, in a test system, in response to such a test agent, the test agent is at a concentration sufficient to cause a measurable biological parameter, referred to as the "EC" of the test agent, to be reduced by 30% of the maximum level of such a measurable biological parameter 30 ". Similarly, the term "EC 100 "is used to denote the effective concentration of a reagent that results in a measurable parameter that produces a maximum (100%) response to the reagent. Similarly, the term EC 50 By (which is generally used in the field of pharmacokinetics) is meant a concentration of the agent sufficient to cause a half-maximal (about 50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum possible amount of test agent that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard temperature and pressure conditions. In pharmacokinetics, the saturation concentration of a drug is generally used to represent the concentration of the drug sufficient to cause all available receptors to be occupied by the drug, while EC 50 Is the concentration of drug that gives half maximal effect.
Enriched (enriched): the term "enriched" as used herein refers to the non-natural manipulation of a sample such that a substance of interest (e.g., a molecule or cell) is present in the following cases: (a) Higher concentration (e.g., higher concentration) than the substance in the starting sample, e.g., a biological sample (e.g., where the molecule is naturally present or present after administration)At least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold higher). Or (b) at a concentration higher than the environment in which the molecule is prepared (e.g., in a recombinantly modified bacterial or mammalian cell).
Extracellular domain: the term "extracellular domain" or its abbreviation "ECD" as used herein refers to the portion of a cell surface protein (e.g., cell surface receptor) outside the cytoplasmic membrane. The cell surface protein may be a transmembrane protein, a cell surface or a membrane associated protein.
Identity of: the term "identity" as used herein with respect to a polypeptide or DNA sequence refers to subunit sequence identity between two molecules. When a subunit position in two molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), then the two molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the amount of identical positions. Typically, sequences are aligned to obtain the highest order match. The identity can be calculated, if necessary, using published techniques and widely used computer programs, such as the BLAST 2.0 algorithm, described in Altschul et al (1990) J.mol.biol.215:403-410 and Altschul et al (1977) Nucleic Acids Res.25:3389-3402. Software for performing BLAST analysis is publicly available from the national center for biotechnology information (National Center for Biotechnology Information) (NCBI) website. The algorithm comprises the following steps: high scoring sequence pairs (HSPs) are first identified by identifying short words of length W in the query sequence that match or satisfy some positive threshold score "T" when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits are used as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score is raised. For nucleotide sequences, the parameter "M" (reward score for a pair of matching residues; always) >0) And "N" (penalty for mismatched residues; always<0) A cumulative score is calculated. For amino acid sequences, the cumulative score matrix is used to calculate the cumulative scoreScoring. The extension of word hits in various directions is aborted when: (a) the cumulative alignment score is reduced by X from its maximum obtained value; the cumulative score becomes zero or below due to the accumulation of one or more negative scoring residue alignments; or (b) to the end of either sequence. The BLAST algorithm parameters "W", "T" and "X" determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly but with default values as follows: word length ("W") 28, expected value ("E") 10, m=1, n= -2, and comparing the two chains. Regarding amino acid sequences, the BLASTP program uses the following default values: the word length (W) is 3, the expected value (E) is 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) PNAS (USA) 89:10915-10919).
In an amount sufficient to produce a response/reaction/remission (response): the phrase "in an amount sufficient to produce a response/remission (response)" as used herein is intended to refer to an amount of test agent sufficient to provide a detectable change in the level of the measured indicator before (e.g., baseline level) and 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 fluorescence test. In some embodiments, the test system is an in vivo system that involves measuring changes in the level of a parameter of a cell, tissue or organism that reflects a biological function before and after application of the test agent to the cell, tissue or organism. In some embodiments, the indicator reflects the biological function or developmental status of the cells assessed in the assay in response to administration of an amount of the test agent. In some embodiments, the test system involves measuring a change in the level of an indicator of a cell, tissue or organism reflecting a biological function before and after applying one or more test agents to the cell, tissue or organism. The term "sufficient amount to produce a response" may be an amount sufficient to be therapeutically effective, but may also be more or less than a therapeutically effective amount.
Inhibitors: the term "inhibitor" as used herein is a moiety that reduces, blocks, prevents, delays activation, inactivates, desensitizes, or down regulatesA molecule, e.g., a gene, protein, ligand, receptor, or cell. Inhibitors may also be defined as molecules that reduce, block or inactivate the constitutive activity of a cell or organism.
Intracellular domains: the term "intracellular domain" or its abbreviation "ICD" as used herein refers to a cell surface protein moiety (e.g., a cell surface receptor) within the cytoplasmic membrane. ICDs may comprise the entire intracellular portion of a transmembrane protein or membrane associated protein, or an intracellular protein.
Separated from each other: the term "isolated" as used herein refers to a polypeptide of interest, if naturally occurring, in an environment different from that in which it might naturally occur. By "isolated" is meant a polypeptide comprised in a sample that is substantially enriched for the polypeptide of interest and/or the polypeptide of interest is partially or substantially purified. If the polypeptide is not naturally occurring, "isolated" means that the polypeptide has been isolated from its environment by synthesis, for example from a recombinant cell culture comprising cells engineered to express the polypeptide, or by solution from a solid phase synthesis.
Kabat numbering: as used herein, the term "Kabat numbering" is art-recognized and refers to the system of numbering amino acid residues in the heavy and light chain regions of immunoglobulins that are more variable than other amino acid residues (e.g., hypervariable), kabat, et al, (1971) Ann.NY Acad.Sci.190:382-93; kabat, et al, (1991) sequence of proteins of immunological interest (Sequences of Proteins of Immunological Interest), fifth edition, U.S. health and public service, NIH publication No. 91-3242. As used herein, the term "Chothia numbering" is art-recognized and refers to a system of numbering amino acid residues based on the position of structural loop regions (Chothia et al 1986,Science 233:755-758; chothia and Lesk 1987, JMB 196:901-917; chothia et al 1992, JMB 227:799-817). For the purposes of this disclosure, unless otherwise specifically indicated, the positioning of CDRs 2 and 3 in the variable regions of the antibody follows the Kabat numbering or simply "Kabat". The localization of CDR1 in the variable region of the antibody follows a hybrid of the Kabat and Chothia numbering schemes.
Ligand: as used herein, the term "ligand" refers to a molecule that specifically binds to a receptor and causes a change in the receptor, which alters the activity of the receptor or reacts in cells expressing the receptor. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term "ligand" includes both natural and synthetic ligands. "ligand" also includes small molecules, cytokines and peptidomimetics of antibodies. The complex of ligand and receptor is referred to as a "ligand-receptor complex". The ligand may comprise a domain of a polyprotein or fusion protein (e.g., or a domain of an antibody/ligand fusion protein)
Regulation of: as used herein, the terms "modulate", "modulating" and the like refer to the ability of a test agent to elicit a response in a system (including biological systems or biochemical pathways), either positively or negatively, or directly or indirectly. The term modulator includes agonists (including partial agonists, full agonists and superagonists) and antagonists.
Nucleic acid: the terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNAs (mrnas), complementary DNAs (cdnas), recombinant polynucleotides, vectors, probes, primers, and the like.
Operatively connected to: the term "operatively linked" as used herein refers to a relationship between molecules, typically polypeptides or nucleic acids, which are arranged in a construct such that the function of each component molecule is preserved, although operative linkage may result in positive or negative regulation of the activity of the individual components in the construct. For example, the operative attachment of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct in which the biological activity of the protein is reduced relative to the wild-type molecule, but the two are still considered to be operatively attached. When the term "operatively connected" is used When referring to the relationship of multiple nucleic acid sequences encoding different functions when combined into a single nucleic acid molecule, for example, when introduced into a cell using recombinant techniques, a nucleic acid is provided that is capable of effecting transcription and/or translation of the particular nucleic acid sequence in the cell. For example, if it results in expression of a preprotein, the nucleic acid sequence encoding the signal sequence may be considered operably linked to DNA encoding the polypeptide, whereby the signal peptide facilitates secretion of the polypeptide; promoters or enhancers are considered to be operably linked to a coding sequence if it affects the transcription of the sequence; or if it is positioned to facilitate translation, the ribosome binding site is thought to be operatively linked to a coding sequence. Generally, in the context of nucleic acid molecules, the term "operatively linked" means that the linked nucleic acid sequences are contiguous, in the context of the secretory leader or associated subdomain of the molecule, which is contiguous and in reading phase. However, certain genetic elements, such as enhancers, may function at a distance, need not be contiguous with the sequence in which they function, but may be considered to be operably linked.
Parent polypeptide: as used herein, the term "parent polypeptide" or "parent protein" is used interchangeably to refer to a source of a second polypeptide (e.g., 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 form of the protein. In some cases, a parent polypeptide may be modified to form a further modified naturally occurring protein. The term "parent polypeptide" may refer to the polypeptide itself or to a composition comprising the parent polypeptide (e.g., glycosylated or PEGylated form and/or fusion protein comprising the parent polypeptide).
Partial agonists: as used herein, the term "partial agonist" refers to a molecule that specifically binds to and activates a given receptor, but has only a partial activation of the receptor relative to a full agonist. Partial agonists may exhibit both agonism and antagonism. For example, when a full agonist and a partial agonist are present simultaneously, the partial agonist acts as a competitive antagonist, by competing with the full agonist for binding to the receptor, relativeContact of the receptor with the full agonist in the absence of the partial agonist results in a net decrease in receptor activation. Partial agonists in a subject may be used to activate a receptor to provide a desired sub-maximal response when an insufficient amount of endogenous ligand is present, or they may reduce over-stimulation of the receptor when an excessive amount of endogenous ligand is present. Maximum response by partial agonists (E Maximum value ) Referred to as its intrinsic activity, a full agonist can be expressed in percent when it produces a 100% response. Partial agonists may have greater than 10% but less than 100%, or greater than 20% but less than 100%, or greater than 30% but less than 100%, or greater than 40% but less than 100%, or greater than 50% but less than 100%, or greater than 60% but less than 100%, or greater than 70% but less than 100%, or greater than 80% but less than 100%, or greater than 90% but less than 100% of the activity of a reference polypeptide when evaluated at similar concentrations in a given assay system.
Polypeptides: the terms "polypeptide," "peptide," and "protein" as used herein are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include genetically encoded or non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The term polypeptide includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences; fusion proteins having heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins having an amino acid sequence (e.g., a chelating peptide) that facilitates purification; fusion proteins with immunological tag proteins; fusion proteins comprising peptides having immunologically active polypeptide fragments (e.g., antigenic diphtheria or tetanus toxins or toxin fragments), and the like.
Receptor(s): as used herein, the term "receptor" refers to a polypeptide having a domain that specifically binds to a ligand, the binding of which results in a change in at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane associated protein comprising an extracellular domain (ECD) and a membrane associated domain for use in administering E CD anchors to the cell surface. In some embodiments of the cell surface receptor, the receptor is a transmembrane polypeptide comprising an intracellular domain (ICD) and an extracellular domain (ECD) connected by a transmembrane domain (membrane spanning domain), commonly referred to as a transmembrane domain (transmembrane domain) (TM). Binding of cognate ligands to the receptor results in conformational changes in the receptor, thereby producing a measurable biological effect. In some cases, if the receptor is a transmembrane polypeptide comprising ECD, TM, and ICD, ligand binding to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to ligand binding to the ECD. In some embodiments, the receptor is a component of a multicomponent complex to promote intracellular signaling. For example, a ligand may bind to a cell surface receptor that is not associated with any intracellular signaling alone, but promotes the formation of heteromultimers (including heterodimers, heterotrimers, etc.) or homomultimer (e.g., homodimers, homotrimers, homotetramers, etc.) complexes upon ligand binding, resulting in a measurable biological effect in the cell, such as activation of the intracellular signaling cascade (e.g., jak/STAT pathway). In some embodiments, the receptor is a transmembrane single chain 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.
Recombination: as used herein, the term "recombinant" is used as an adjective to refer to a method of modifying a polypeptide, nucleic acid, or cell using recombinant DNA technology. "recombinant protein" refers to a protein produced using recombinant DNA techniques, often abbreviated in the lowercase "r" prior to the protein name, to refer to the method of producing the protein (e.g., recombinantly produced human growth hormone is often abbreviated in the abbreviation "rhGH". Similarly, if the cell is modified, foreign nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.) are incorporated (e.g., transfected, transduced, infected) by using recombinant DNA techniques, the cell is referred to as a "recombinant cell". The techniques and protocols for recombinant DNA techniques are well known in the art, e.g., can be found in Sambrook et al (1989) Molecular cloning: laboratory Manual (Molecular Manual)Cloning A Laboratory Manual) (2 nd edition, cold spring harbor laboratory Press, prain, N.Y.) and other standard molecular biology laboratory manuals.
Response/alleviation of: the term "response/alleviation", for example, a response of a cell, tissue, organ or organism, includes a quantitative or qualitative change in an assessed biochemical or physiological parameter (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy expenditure rate (rate of energy consumption), differentiation level or state), wherein the change is associated with activation, stimulation or treatment, or with contact by an exogenous agent or an internal mechanism (e.g., genetic programming). In some cases, the terms "activate", "stimulus" and the like refer to cellular activation regulated by internal mechanisms, external or environmental factors; whereas the terms "inhibit", "down" and the like refer to the opposite effect. "response/alleviation" can be assessed in vitro, for example by using assay systems, surface plasmon resonance, enzyme activity, mass spectrometry, amino acid or protein sequencing techniques. "response/remission" can be quantitatively assessed in vivo, by assessing objective physiological parameters such as body temperature, body weight, tumor volume, blood pressure, the results of X-ray or other imaging techniques, or by reporting changes in subjective feelings (happiness, depression, anxiety or pain). In some embodiments, the proliferation level of CD3 activated primary human T cells can be assessed in a bioluminescence assay that produces a luminescent signal proportional to the amount of ATP present, which is proportional to the amount of viable cells present in the culture, as described by Crouch, et al (1993) J.Immunol. Methods 160:81-8, or using commercially available assays, for example 2.0 cell viability assay or +.>3D cell viability kit commercially available from Promega (Promega Corporation), madison WI 53711, cat# G9241 andg9681, substantially according to the instructions provided by the manufacturer. In some embodiments, the level of activation of T cells in response to administration of a test agent can be determined by the flow cytometry method, as determined by STAT (e.g., STAT1, STAT3, STAT 5) phosphorylation levels, according to methods well known in the art.
Significantly reduced binding:as used herein, the term "exhibiting significantly reduced binding" is used in that a variant of a first molecule (e.g., a ligand or antibody) exhibits significantly reduced affinity for a second molecule (e.g., a receptor or antigen) as compared to the parent form of the first molecule. An antibody variant "exhibits significantly reduced binding" for an antibody variant if the binding of the variant to the native form of the receptor is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5%, of the parent antibody from which the variant is derived. Similarly, a variant ligand "exhibits significantly reduced binding" for the variant ligand if the affinity of the variant ligand to bind to the receptor is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5%, of the affinity of the parent ligand from which the variant ligand is derived. Similarly, a variant ligand "exhibits significantly reduced binding" for a variant receptor if the variant receptor binds with an affinity that is less than 20%, or less than about 10%, or less than about 8%, or less than about 6%, or less than about 4%, or less than about 2%, or less than about 1%, or less than about 0.5% of the parent receptor from which the variant receptor is derived.
One or more small molecules: the term "small molecule" refers to chemical compounds (typically pharmaceutically active compounds) small molecules having a molecular weight of less than about 10kDa, less than about 2kDa, or less than about 1kDa, including, but not limited to, inorganic molecules, organic molecules comprising inorganic components, molecules comprising radioactive atoms, and synthetic molecules. The term "small molecule" is a term well known to those of ordinary skill in the pharmaceutical industry and is commonly used to distinguish between organic compoundsChemical compounds and biological agents.
Specific binding:the term "specific binding" as used herein refers to the degree to which a first molecule exhibits affinity/avidity for a second molecule. In the context of a binding pair (e.g., ligand/receptor, antibody/antigen), a first molecule of the binding pair is said to specifically bind to a second molecule of the binding pair when the first molecule does not bind to other components present in the sample in significant amounts. 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 greater, or at least 5-fold greater, or at least 10-fold greater, or at least 20-fold greater, or at least 100-fold greater than the affinity of the first molecule for other components present in the sample. In particular embodiments in which the first molecule in the binding pair is an antibody, if the equilibrium dissociation constant (K D ) Greater than about 10 6 M, or greater than about 10 8 M, or greater than about 10 10 M, or greater than about 10 11 M is greater than about 10 12 M, as determined, for example, by Scatchard analysis, the antibody specifically binds to an antigen (or an epitope of a protein, antigen, ligand or receptor) (Munsen, et al (1980) analysis. Biochem. 107:220-239). In one embodiment, wherein the ligand is an sdAb that binds IL27 ra, the receptor comprises IL27 ra, if the balance dissociation constant of the sdAb/IL27 ra ECD that binds IL27 ra is greater than about 10 5 M, or greater than about 10 6 M, or greater than about 10 7 M, or greater than about 10 8 M, or greater than about 10 9 M, or greater than about 10 10 M, or greater than about 10 11 M, sdAb binding IL27 ra specifically binds. Specific binding can be assessed using techniques known in the art, including, but not limited to, competition ELISA, radioligand binding assays (e.g., saturated binding, scatchard plot (Scatchard plot), non-linear curve fitting programs, and competition binding assays); nonradioactive ligand binding assays (e.g., fluorescence polarization (fluorescence polarization) (FP), fluorescence Resonance Energy Transfer (FRET), liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation) ) The method comprises the steps of carrying out a first treatment on the surface of the In some embodiments, the invention provides molecules that specifically bind to IL27Rα isoforms (e.g., sdabs that bind IL27Rα) as used herein, the binding affinity of IL27Rα binding molecules to IL27Rα can be determined and/or quantified by surface plasmon resonance ("SPR") using commercially available instruments such as Biacore 8K, biacore 8K+, biacore S200, biacore T200 (Cytiva, 100Results Way, markler 01752, massachusetts.) in evaluating the binding affinity of IL27Rα binding molecules to IL27Rα, in some embodiments, a sensor chip on which a protein of interest is to be immobilized is coupled to a substance that facilitates binding of the protein of interest, such as a nitrilotriacetic acid (NTA) -derived surface plasmon resonance sensor chip (e.g., a sensor chip NTA available from the global life sciences solution, america, inc. (Cytiva Global Life Science Solutions USA LLC), markerle, massachusetts, catalog No. BR 100407), as an anti-His tag antibody (e.g., an anti-histidine CM5 chip commercially available from Cytiva, markerle, massachusetts), protein a, or biotin), thus, in order to evaluate binding, it is often desirable to modify the protein to provide binding to the substance coupled to the chip surface, one member of the binding pair is evaluated by incorporating a chelating peptide comprising a polyhistidine sequence (e.g., 6xHis or 8 xHis) to remain on the chip coupled to NTA. In some embodiments, the IL27 ra binding molecule may be immobilized on a chip, and the IL27 ra (or ECD fragment thereof) may be provided in the mobile phase. Alternatively, the IL27 ra (or ECD fragment thereof) may be immobilized on a chip, and the IL27 ra binding molecule may be provided in the mobile phase. In either case, it should be noted that modification of some of the proteins immobilized on the coated SPR chip may interfere with the binding properties of one or both components of the binding pair to be evaluated by SPR. In this case, it may be necessary to switch the mobility of the binding pair And binding elements, or using chips with binding agents that promote non-interfering coupling of the proteins to be evaluated. Alternatively, when SPR is used to assess the binding affinity of an IL27Rα binding molecule for IL27Rα, the IL2Rb binding molecule may be derivatized by C-terminal addition of a poly-His sequence (e.g., 6XHis or 8 XHis) and immobilized on an NTA-derivatized sensor chip, providing in the mobile phase the IL27Rα receptor subunit whose ligand is being assessed for binding affinity. Methods for incorporating poly-His sequences into the C-terminus of IL27Rα binding molecules produced by recombinant DNA techniques are well known to those skilled in the art of biotechnology. In some embodiments, the binding affinity of an IL27 ra binding molecule using SPR for IL27 ra substantially conforms to the teachings of the examples.
Object(s): the terms "subject," "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, and the like. In some embodiments, the mammal is a human.
Substantially pure: as used herein, the term "substantially pure" means that the components of the composition comprise greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95% of the total composition. The "substantially pure" protein comprises greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95% of the total composition.
T-cell:the term "T-cell" or "T-cell" as used herein is used in its conventional sense to refer to a lymphocyte that differentiates in the thymus, possesses a specific cell surface antigen receptor, and includes some lymphocytes that control initiation or suppression of cell-mediated and humoral immunity and other lymphocytes that lyse antigen-bearing cells. In some embodiments of the present invention, in some embodiments,t cells include, but are not limited to, naive CD8 + T cell, 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 The method comprises the steps of carrying out a first treatment on the surface of the Regulatory T cells, e.g. T R 1. Treg, inducible Treg; 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 cell is a T cell expressing an IL27rα isoform, interchangeably termed an IL27rα cell, an IL27rα+ cell, an IL27rα T cell, or an IL27rα+ T cell.
End to end: as used herein in the context of polypeptide structure, "N-terminal" (or "amino-terminal") and "C-terminal" (or "carboxy-terminal") refer to the extreme amino-and carboxy-terminal, respectively, of a polypeptide, while the terms "N-terminal" and "C-terminal" refer to the relative positions in the amino acid sequence of a polypeptide toward the N-terminal and C-terminal, respectively, and may include the N-terminal and C-terminal residues, respectively. "N-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue in a continuous polypeptide sequence, the first amino acid being closer to the N-terminus of the polypeptide. "close proximity" to the C-terminus refers to the position of a first amino acid residue relative to a second amino acid residue in a continuous polypeptide sequence, the first amino acid being closer to the C-terminus of the polypeptide.
Transmembrane domain: the term "transmembrane domain" or "TM" refers to a polypeptide domain of a transmembrane polypeptide (e.g., a transmembrane receptor) that, when associated with a cell membrane, is embedded in the cell membrane and linked to the extracellular domain (ECD) and intracellular domain (ICD) of the transmembrane polypeptide by a peptidyl linkage. The transmembrane domain may be homologous (naturally associated) or heterologous (not naturally associated) to one or both of the extracellular and/or intracellular domains. In some embodiments, when the receptor is a chimeric receptor comprising 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 the ICD or ECD of the parent receptor from which the chimeric receptor is derived.
Treatment/management: the terms "treat," "treatment," and the like refer to, corresponding to a diagnosis of a subject having a disease, disorder, or condition, or symptom thereof, initiating a course of action for the subject (e.g., contacting the subject with a pharmaceutical composition comprising an sdAb that binds IL27 ra, alone or in combination with a supplement) to temporarily or permanently eliminate, reduce, inhibit, reduce, or ameliorate at least one of: (a) The root cause of a disease, disorder or condition afflicting a subject; and/or (b) at least one symptom associated with such a disease, disorder or condition. In some embodiments, the treatment comprises a course of action taken on a subject having a disease, wherein the course of action results in the subject's disease being inhibited (e.g., preventing the development of a disease, disorder, or condition or ameliorating one or more symptoms associated therewith).
Treg cells or regulatory T cells. The terms "regulatory T cells", "Treg cells" or "Treg" interchangeably herein refer to CD4 + Types of T cells capable of inhibiting other T cell responses, including but not limited to effector T cells (T eff ). Treg cells are generally characterized by expression of CD4 (cd4+), the CD25 subunit of the IL2 receptor (cd25+) and the transcription factor fork box P3 (foxp3+) (Sakaguchi, annu Rev Immunol 22,531-62 (2004). In some cases, the term "conventional CD4 + T cells "are used to differentiate non-Treg CD4 + T cells and CD4 + Treg。
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 may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified form of a WT polypeptide. The term variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding the same. In some embodiments, the variant polypeptide comprises from about 1 to about 1 as compared to the parent polypeptideAbout 10, or about 1 to about 8, or about 1 to about 7, or about 1 to about 5, or about 1 to about 4, or about 1 to about 3, or 1 to 2 amino acid modifications, substitutions or deletions, or single amino acid modifications, substitutions or deletions. A variant may be at least about 99% identical, or at least about 98% identical, or at least about 97% identical, or at least about 95% identical, or at least about 90% identical to the parent polypeptide from which it is derived.
Wild type:"wild-type" or "WT" or "native" refers herein to an amino acid sequence or nucleotide sequence that occurs in nature, including allelic variation. Wild-type proteins, polypeptides, antibodies, immunoglobulins, igG, etc., have amino acid sequences or nucleotide sequences that are not artificially modified.
IL27Rα
The IL27Rα binding molecules of the invention specifically bind to the extracellular domain of IL27Rα.
Human IL27Rα
In one embodiment, specifically binds to the extracellular domain of the human IL27 ra receptor subunit (hll 27 ra). hIL27Rα is expressed as a 636 amino acid precursor comprising a 32 amino acid N-terminal signal sequence that is cleaved post-translationally to provide a 604 amino acid mature protein. The classical full-length acid hIL27 ra precursor (comprising a signal peptide) is a 636 amino acid polypeptide having the amino acid sequence:
MRGGRGAPFWLWPLPKLALLPLLWVLFQRTRPQGSAGPLQCYGVGPLGDLNCSWEPLGDLGAPSELHLQSQKYRSNKTQTVAVAAGRSWVAIPREQLTMSDKLLVWGTKAGQPLWPPVFVNLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKVLICQFHYRRCQEAAWTLLEPELKTIPLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSGNLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPRSVAVSSIAGSTELLVTWQPGPGEPLEHVVDWARDGDPLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAPLVGPTLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQSGTSPSVCMNVSGNTQSVTLPDLPWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWKVLPGILFLWGLFLLGCGLSLATSGRCYHLRHKVLPRWVWEKVPDPANSSSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESSQPAQATAPLDSGYEKHFLPTPEELGLLGPPRPQVLA
(SEQ ID NO:1)
for the purposes of the present invention, numbering of the amino acid residues of the human IL27Rα polypeptide as described herein is performed according to the numbering of the classical sequence (UniProt reference Q6UWB1, SEQ ID NO: 1). Amino acids 1-32 of SEQ ID NO. 1 are identified as signal peptides of hIL27R alpha, amino acids 33-516 of SEQ ID NO. 1 are identified as extracellular domains, amino acids 517-537 of SEQ ID NO. 1 are identified as transmembrane domains, and amino acids 538-636 of SEQ ID NO. 1 are identified as intracellular domains.
For the purpose of generating antibodies that bind to the ECD of IL27 ra, immunization can be performed with the extracellular domain of hll 27 ra. The extracellular domain of hll 27 ra is a 484 amino acid polypeptide of the sequence:
QGSAGPLQCYGVGPLGDLNCSWEPLGDLGAPSELHLQSQKYRSNKTQTVAVAAGRSWVAIPREQLTMSDKLLVWGTKAGQPLWPPVFVNLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKVLICQFHYRRCQEAAWTLLEPELKTIPLTPVEIQDLELATGYKVYGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSGNLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEGITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPRSVAVSSIAGSTELLVTWQPGPGEPLEHVVDWARDGDPLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLASASSVWGFREELAPLVGPTLWRLQDAPPGTPAIAWGEVPRHQLRGHLTHYTLCAQSGTSPSVCMNVSGNTQSVTLPDLPWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWK
(SEQ ID NO:192).
mouse IL27Rα
In one embodiment, specifically binds to the extracellular domain of the mouse or murine IL27 ra receptor subunit (mll 27 ra). The mIL27Rα is expressed as a 623 amino acid precursor comprising a 24 amino acid N-terminal signal sequence that is cleaved post-translationally to provide a 599 amino acid mature protein. The classical full-length acid mIL27Rα precursor (signal peptide comprising 24 amino acids) is a 623 amino acid polypeptide having the amino acid sequence:
MNRLRVARLTPLELLLSLMSLLLGTRPHGSPGPLQCYSVGPLGILNCSWEPLGDLETPPVLYHQSQKYHPNRVWEVKVPSKQSWVTIPREQFTMADKLLIWGTQKGRPLWSSVSVNLETQMKPDTPQIFSQVDISEEATLEATVQWAPPVWPPQKVLICQFRYKECQAETWTRLEPQLKTDGLTPVEMQNLEPGTCYQVSGRCQVENGYPWGEWSSPLSFQTPFLDPEDVWVSGTVCETSGKRAALLVWKDPRPCVQVTYTVWFGAGDITTTQEEVPCCKSPVPAWMEWAVVSPGNSTSWVPPTNLSLVCLAPESAPCDVGVSSADGSPGIKVTWKQGTRKPLEYVVDWAQDGDSLDKLNWTRLPPGNLSTLLPGEFKGGVPYRITVTAVYSGGLAAAPSVWGFREELVPLAGPAVWRLPDDPPGTPVVAWGEVPRHQLRGQATHYTFCIQSRGLSTVCRNVSSQTQTATLPNLHLGSFKLWVTVSTVAGQGPPGPNLSLHLPDNRIRWKALPWFLSLWGLLLMGCGLSLASTRCLQARCLHWRHKLLPQWIWERVPDPANSNSGQPYIKEVSLPQPPKDGPILEVEEVELQPVVESPKASAPIYSGYEKHFLPTPEELGLLV
(SEQ ID NO:193)
for the purposes of the present invention, numbering of amino acid residues of the mIL27Rα polypeptide as described herein is performed according to the numbering of the classical sequence (UniProt reference number O70394, SEQ ID NO: 193). Amino acids 1-24 of SEQ ID NO:193 are identified as signal peptides of mIL27Rα, amino acids 23-510 of SEQ ID NO:193 are identified as extracellular domains, amino acids 511-531 of SEQ ID NO:193 are identified as transmembrane domains, and amino acids 532-623 of SEQ ID NO:193 are identified as intracellular domains.
For the purpose of generating antibodies that bind to the ECD of IL27 ra, immunization can be performed with the extracellular domain of ml27 ra. The extracellular domain of the mll 27rα receptor is a 486 amino acid polypeptide, the sequence of which is:
TRPHGSPGPLQCYSVGPLGILNCSWEPLGDLETPPVLYHQSQKYHPNRVWEVKVPSKQSWVTIPREQFTMADKLLIWGTQKGRPLWSSVSVNLETQMKPDTPQIFSQVDISEEATLEATVQWAPPVWPPQKVLICQFRYKECQAETWTRLEPQLKTDGLTPVEMQNLEPGTCYQVSGRCQVENGYPWGEWSSPLSFQTPFLDPEDVWVSGTVCETSGKRAALLVWKDPRPCVQVTYTVWFGAGDITTTQEEVPCCKSPVPAWMEWAVVSPGNSTSWVPPTNLSLVCLAPESAPCDVGVSSADGSPGIKVTWKQGTRKPLEYVVDWAQDGDSLDKLNWTRLPPGNLSTLLPGEFKGGVPYRITVTAVYSGGLAAAPSVWGFREELVPLAGPAVWRLPDDPPGTPVVAWGEVPRHQLRGQATHYTFCIQSRGLSTVCRNVSSQTQTATLPNLHLGSFKLWVTVSTVAGQGPPGPNLSLHLPDNRIRWK
(SEQ ID NO:194)
IL27Rα binding molecules and single domain antibodies
In some embodiments, the IL27 ra binding molecules of the invention are single domain antibodies (sdabs). The present invention relates to IL27 ra binding molecules comprising single domain antibodies (sdabs) that specifically bind to the extracellular domain of a human IL27 ra isoform (hIL 27 ra), which are found in all IL27 ra expressing cells.
Single domain antibodies (sdabs) are antibodies that comprise a single monomer variable antibody domain. Like full-length antibodies, sdabs are capable of specifically binding to an epitope. VHH single domain antibodies that bind hll 27 ra may be engineered from heavy chain antibodies isolated from camelidae mammals (e.g., camels, llamas, dromedaries, alpacas, and camelos) immunized with the hll 27 ra extracellular domain or immunologically active fragments thereof. Descriptions of sdabs and VHHs can be 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 (Antibodies)(Basel) 8 (1); and De Vlieger, et al (2018)Anti-cancer Body (Antibodies)(Basel) 8 (1). Alternatively, the 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. The sdAb that binds to hll 27 ra can also be obtained by cleaving dimer variable domains from immunoglobulin G (IgG) isotypes from other mammalian species including humans, rats, rabbits immunized with hll 27 ra extracellular domains or immunologically active fragments thereof. While most current studies on sdabs are based on heavy chain variable domains, sdabs from the light chain have also been demonstrated to specifically bind to target proteins comprising an antigen immune sequence. Moller et al, JBiol chem.285 (49): 38348-38361,2010.
In some embodiments, the sdAb is a VHH. VHH is an sdAb with a single heavy chain variable antibody domain. Similar to traditional antibodies, VHH is capable of specifically binding to a specific antigen. Exemplary VHHs have a molecular weight of about 12-15kDa, which is much smaller than conventional mammalian antibodies (150-160 kDa) that are composed of two heavy chains and two light chains. VHH can be found or produced in camelidae mammals (e.g. camels, llamas, dromedaries, alpacas and camelos) which naturally lack light chains.
The present invention provides IL27Rα binding molecules comprising polypeptides having at least 75%, or 80%, or 90%, or 95%, or 98%, or 99%, or 100% identity relative to any one of the polypeptides of SEQ ID NOs 2-25.
The present invention provides IL27Rα binding molecules comprising polypeptides having at least 75%, or 80%, or 90%, or 95%, or 98%, or 99%, or 100% identity relative to any one of the polypeptides of SEQ ID NOS: 61-74.
The present invention provides IL27 ra binding molecules comprising CDR1, CDR2 and CDR3 as set forth in the rows 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, or have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, relative to the sequences described in the rows of table 1 provided herein.
The present invention provides IL27 ra binding molecules comprising CDR1, CDR2 and CDR3 as set forth in the rows 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, or have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, relative to the sequences described in the rows of table 3 provided herein.
Experiment
The single domain antibodies of the invention are obtained from camels by immunization with the extracellular domain of the IL27 ra receptor. The IL27 ra VHH molecules of the invention are substantially produced according to the teachings of the examples. Briefly, a camel is sequentially immunized with ECD of human IL27 ra and mouse IL27 ra by subcutaneous injection of an adjuvant composition containing a recombinantly produced fusion protein comprising the extracellular domain of IL27 ra, the human IgG1 hinge domain, and the human IgG1 heavy chain Fc over a period of several weeks. Following immunization, RNA extracted from a blood sample of a suitably sized VHH-hinge-CH 2-CH3 species is transcribed to produce a DNA sequence which is digested to identify an isolated about 400bp fragment comprising a nucleic acid sequence encoding a VHH domain. The isolated sequences were digested with restriction enzymes to facilitate insertion into phage vectors, in frame with the sequences encoding his-tags, and transformed into E.coli to generate phage libraries. Phage libraries were subjected to multiple rounds of biopanning (bio-panning) to identify VHHs (human or mouse, as the case may be) that bind to the ECD of IL27 ra. Individual phage clones were isolated in 96-well plates for periplasmic extract ELISA (PE-ELISA) and selective binding was confirmed by colorimetric assay. IL27Rα binding molecules that exhibit specific binding to IL27Rα antigen are isolated and sequenced, and the sequences are analyzed to identify VHH sequences, CDRs, and to identify unique VHH clonotypes. As used herein, the term "clonotype" refers to a collection of binding molecules derived from the same B cell progenitor cell, in particular a collection of antigen binding molecules belonging to the same germline family, having the same CDR3 length and having 70% or more homology in the CDR3 sequence. VHH molecules (anti-human IL27rα VHH) and CDRs isolated from these VHHs that show specific binding to the hll 27rα ECD antigen are provided in table 1. VHH molecules (anti-mouse IL27rα VHH) and CDRs isolated from these VHHs that show specific binding to the ml27 rα ECD antigen are provided in table 3. Tables 2 and 4 provide the nucleic acid sequences encoding VHHs of tables 1 and 3, respectively.
In order to more fully characterize the binding properties and evaluate the binding affinity of VHH molecules generated according to the above, representative examples of each human VHH clonotype were subjected to surface plasmon resonance analysis, substantially in accordance with the teachings of example 5 herein. The results of these SPR studies are summarized in table 6 below.
/>
* When R is Maximum value >At 100, both association and dissociation kinetic constants may be inhibited. This effect, if present, is likely to be offset in the kinetic ratio, i.e. the affinity constant.
As shown in the data presented in table 6, the IL27 ra VHH binding molecules described above exhibit specific binding to an antigen and provide a range of affinities for the IL27 ra antigen.
In some cases, immunization with an antigen derived from IL27 ra of a first mammalian species (e.g., hIL27 ra-ECD) can provide antibodies that specifically bind to IL27 ra receptors of one or more other mammalian species due to sequence or structural similarity between the extracellular domains of IL27 ra receptors from the various mammalian species. Such antibodies are referred to as "cross-reactive". For example, immunization of camels with human antigens (e.g., hIL27Rα -ECD) may produce antibodies that cross-react with murine and human receptors. For the assessment of antibody cross-reactivity of receptors derived from other mammalian species, one skilled in the art can readily determine, for example, using methods related to the assessment of binding affinity and/or specific binding as described elsewhere herein, such as flow cytometry or SPR. Thus, the use of the term "human IL27RαVHH" or "hIL27RαVHH" merely indicates that the species of IL27Rα antigen used to immunize a VHH-derived camel is human IL27Rα (e.g., hIL27Rα, ECD, SEQ ID NO: 192), but should not be construed as limiting the specific binding affinity of the VHH to hIL27Rα molecules of other mammalian species. Similarly, the use of the term "mouse IL27RαVHH" or "mIL27Rα" merely indicates that the species of IL27Rα antigen used to immunize a VHH-derived camel is murine IL27Rα (e.g., mIL27Rα, ECD, SEQ ID NO: 194), but should not be construed as limiting the specific binding affinity of the VHH to IL27Rα molecules of other mammalian species.
Modification of single domain antibodies
CDR-grafted sdAb
In some embodiments, an sdAb of the invention that binds IL27 ra is a CDR-grafted sdAb that binds IL27 ra. CDRs obtained from antibodies, heavy chain antibodies and sdabs derived therefrom can be grafted onto alternative frameworks to produce CDR-grafted sdabs, as described in Saerens et al (2005) j.mol Biol 352:597-607. In some embodiments, the invention provides an IL27 ra binding molecule comprising a CDR-grafted IL27 ra-binding sdAb comprising a set of CDRs 1, 2, and 3, as indicated in the rows of table 3 above.
Chimeric and humanized sdabs
Any framework region can be used with the CDRs described herein. In some embodiments, the sdAb that binds IL27 ra is a chimeric sdAb, wherein the CDRs are derived from one species (e.g., camelid) and the framework and/or constant regions are derived from another species (e.g., human or mouse). In a particular embodiment, the framework region is a human or humanized sequence. Thus, humanized IL27Rα -binding sdabs derived from IL27Rα -binding VHHs are considered to be within the scope of the invention. Humanized techniques for camelid single domain antibodies are well known in the art. See, e.g., vincke, et al (2009) [ general strategy for humanization of camelid single domain antibodies and identification of universal humanized nanobody scaffolds (General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold) [ biol. Chem.284 (5) 3273-3284 ].
In some embodiments, V described herein H H may be humanized to comprise human framework regions. Can be used for preparing humanized V H Examples of human germline of H include, but are not limited to, VH3-23 (e.g., uniProt ID: P01764), VH3-74 (e.g., uniProt ID: A0A0B4J1X 5), VH3-66 (e.g., uniProt ID: A0A0C4DH 42), VH3-30 (e.g., uniProt ID: P01768), VH3-11 (e.g., uniProt ID: P01762), and VH3-9 (e.g., uniProt ID: P01782).
IL27Rα binding molecules comprising other agents
In some embodiments, the IL27 ra binding molecules of the invention comprise an IL27 ra single domain antibody (sdAb) coupled to one or more other bioactive agents, including but not limited to therapeutic agents, chemical, optical, or radioactive agents, including combinations thereof. The coupling of at least one such biological, chemical, optical, or radioactive agent imparts additional biological or chemical properties to the sdAb that binds IL27 ra, and the combination provides IL27 ra binding molecules with additional or alternative uses.
For example, the other agent may be one or more molecules selected from the group consisting of: immunomodulators (e.g., immunogens); molecules that improve water solubility (e.g., water-soluble polymers and hydrophilic molecules such as sugars); carrier molecules that extend in vivo half-life (e.g., pegylation, fc fusion, or acylation); generating antibodies (e.g., epitope tags) for detection assays, improving ease of purification (e.g., chelating peptides, such as poly-His tags); providing a targeting domain that selectively targets an IL27 ra binding molecule to a particular cell or tissue type; therapeutic agents (e.g., therapeutic agents including small molecules or polypeptide agents); reagents (e.g., radionucleotides or fluorescent reagents) that are visible to an optical or electromagnetic sensor. In some embodiments, the linker is a cleavable linker or a non-cleavable linker. The use of a cleavable linker in an IL27 ra binding molecule as contemplated herein facilitates release of the therapeutic agent into the intracellular cytoplasm upon internalization of the IL27 ra binding molecule. The non-cleavable linker will allow release upon digestion of the IL27 ra binding molecule, or the IL27 ra binding molecule may be used with an agent (e.g., imaging agent) that does not require release from the antibody.
In some embodiments, wherein the IL27 ra binding molecule comprises an sdAb that binds IL27 ra that is stably associated with other agents that are linked via a linker. The linker is a covalent link between two elements of the IL27 ra binding molecule (e.g., VHH and PEG polymers that bind hll 27 ra). The linker may be a covalent bond, a chemical linker or a peptide linker. Suitable linkers include "flexible linkers" that are generally of a length sufficient to allow some movement between the sdAb that binds IL27 ra and one or more attached agents. Examples of chemical linkers include arylacetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. In some embodiments, the linker is a peptide linker. Suitable peptide linkers can be readily selected and can be any suitable length, for example 1 amino acid (e.g., gly), 2, 3, 4, 5, 6, 7, 8, 9, 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 (G) n Glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and therefore can be used as neutral linkages between components (tether). Other examples of flexible linkers include glycine polymers (G ) n Glycine-alanine polymer, alanine-serine polymer, glycine-serine polymer. Glycine and glycine-serine polymers are relatively unstructured and therefore can be used as neutral linkages between components (tether). Multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-20, 20-30, or 30-50) of these linker sequences can be linked together to provide flexible linkers that can be used to couple heterologous amino acid sequences to the IL27Rα -binding sdabs disclosed herein. In some embodiments, the linker has the formula (GGGS) n, (GGGSG) n, (GGGGS) n, (GGS) nG 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.
Immunomodulators
In some embodiments, the IL27 ra binding molecules of the invention are operatively linked to an immunomodulatory agent (immunoconjugate). Immunomodulatory agents that can be coupled to a sdAb of the invention that binds hll 27 ra include, but are not limited to, inactivated viral particles, inactivated bacterial toxins such as diphtheria toxoid, tetanus, cholera or leukotoxin molecules, inactivated bacteria, and dendritic cells. Such immunoconjugates may be used to promote an immune response against IL27 ra or cells expressing IL27 ra.
Flag label
In some embodiments, the IL27 ra binding molecules of the invention are operably linked to an antigen tag, such as a FLAG sequence. As described herein, FLAG sequences are recognized by biotinylated, highly specific anti-FLAG antibodies (see, e.g., blanir et al (1992) Science 256:1014 and LeClair et al (1992) PNAS-USA 89:8145). In some embodiments, the sdAb polypeptide that binds IL27 ra further comprises a C-myc epitope tag at the C-terminus.
Chelating peptides
In some embodiments, the IL27 ra binding molecules of the invention are operably linked to one or more transition metal chelating polypeptide sequences. Incorporation of such transition metal chelating domains facilitates purification of Immobilized Metal Affinity Chromatography (IMAC) as described in U.S. Pat. No. 4,569,794 issued to Smith et al, 1986, 2, 11. Transition metal chelate polypeptides useful in the practice of the present IL27Rα binding moleculesExamples of peptides are described in Smith et al (supra) and U.S. patent No. 5,320,663 issued to Dobeli et al, 5/10/1995, the entire teachings of which are incorporated herein by reference. Specific transition metal chelating polypeptides useful in the practice of the present IL27Rα binding molecules are polypeptides comprising 3-6 consecutive histidine residues, such as 6-histidine (His) 6 Peptides are often referred to in the art as "His-tags". In addition to providing a purified "handle" for recombinant proteins or facilitating immobilization on SPR sensor chips, this coupling of h IL27Rα binding molecules to chelating peptides also facilitates targeted delivery of transition metal ions as kinetically inert or kinetically unstable complexes to IL27Rα -expressing cells, essentially in accordance with the teachings of Anderson et al (U.S. Pat. No. 5,439,829 and Hale, J.E (1996) Analytical Biochemistry (1): 46-49. Transition metal ions are reporter molecules, such as fluorescent compounds or radioactive agents, including radiological imaging agents or therapeutic agents, issued 8.8.1995).
Carrier molecules
In some embodiments, an sdAb of the invention that binds IL27 ra can be conjugated to one or more carrier molecules. Carrier molecules are typically large, slowly metabolized macromolecules that provide in vivo stability and/or extended duration of action to distinguish these molecules from conventional carrier molecules used in the preparation of pharmaceutical formulations, as described below. Examples of in vivo carriers that can incorporate IL27 ra binding molecules, but are not limited to: proteins (including but not limited to human serum albumin); fatty acids (acylated); polysaccharides (including, but not limited to, (N-and O-linked) sugars, agarose (sepharose), agarose (agarsose), cellulose or cellulose); polypeptide amino acid copolymers; acylated, or polysialized, polyethylene glycol (PEG) polymers.
Water-soluble polymers
In some embodiments, the sdAb that binds IL27 ra is coupled to one or more water-soluble polymers. Examples of water-soluble polymers useful in the practice of the present IL27 ra binding molecules include polyethylene glycol (PEG), polypropylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly (oxyethylated polyols), polyolefin alcohols (polyolefinic alcohol), polysaccharides, poly-a-hydroxy acids, polyvinyl alcohol (PVA), polyphosphazenes, polyoxazolines (POZ), poly (N-acryloylmorpholines), or combinations thereof.
Polyethylene glycol
In one embodiment, the carrier molecule is a polyethylene glycol ("PEG") polymer. Coupling of PEG polymers to proteins (pegylation) is a well-established method to extend the serum half-life of biological agents. PEGylated polypeptides may also be referred to as mono-, di-, tri-, etc. PEGylated polypeptides to denote polypeptides comprising one, two, three (or more) PEG moieties attached to the polypeptide, respectively. In some embodiments, the PEG can be covalently attached directly to the sdAb (e.g., via a lysine side chain, a sulfhydryl group of a cysteine, or an N-terminal amine) or optionally employ a linker between the PEG and the sdAb. In some embodiments, the IL27 ra binding molecule comprises more than one PEG molecule, each PEG molecule attached to a different amino acid residue. In some embodiments, sdabs can be modified by incorporating unnatural amino acids with non-naturally occurring amino acid side chains to facilitate site-specific pegylation. In other embodiments, the cysteine residue may be substituted at one or more positions within the sdAb to facilitate site-specific pegylation via the cysteine thiol side chain.
In certain instances, the IL27Rα binding molecules of the invention have an N-terminal glutamine ("1Q") residue. Spontaneous cyclization of the N-terminal glutamine residue to form pyroglutamic acid (pE) has been observed under physiological or near physiological conditions. (see, e.g., liu, et al (2011) J.biol. Chem.286 (13): 11211-11217). In some embodiments, the formation of pyroglutamic acid complicates N-terminal PEG coupling, particularly when aldehyde chemistry is used for N-terminal pegylation. Thus, when pegylating the IL27 ra binding molecules of the invention, particularly when aldehyde chemistry is used, the IL27 ra binding molecule having an amino acid at position 1 (e.g., 1Q) is substituted with a substitute amino acid at position 1 or deleted at position 1 (e.g., des-1Q). In some embodiments, the IL27 ra binding molecules of the invention comprise amino acid substitutions selected from the group consisting of Q1E and Q1D.
PEG suitable for coupling to polypeptide sequences is generally soluble in water at room temperature and has the general formula
R(O-CH 2 -CH 2 ) n O-R,
Wherein R is hydrogen or a protecting group, such as an alkyl or alkanol group, wherein n is an integer from 1 to 1000. When R is a protecting group, it typically has 1 to 8 carbons. PEG may be linear or branched. Branched PEG derivatives, "star-PEG" and multi-arm PEG are contemplated by the present invention.
The molecular weight of the PEG used in the IL27 ra binding molecule is not limited to any particular range. The PEG component of the IL27Rα binding molecule can have a molecular weight of greater than about 5kDa, greater than about 10kDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is from about 5kDa to about 10kDa, from about 5kDa to about 15kDa, from about 5kDa to about 20kDa, from about 10kDa to about 15kDa, from about 10kDa to about 20kDa, from about 10kDa to about 25kDa, or from about 10kDa to about 30kDa. The molecular weight of the linear or branched PEG molecule is from about 2,000 to about 80,000 daltons, alternatively from about 2,000 to about 70,000 daltons, alternatively from about 5,000 to about 50,000 daltons, alternatively from about 10,000 to about 50,000 daltons, alternatively from about 20,000 to about 50,000 daltons, alternatively from about 30,000 to about 50,000 daltons, alternatively from about 20,000 to about 40,000 daltons, alternatively from about 30,000 to about 40,000 daltons. In one embodiment of the IL27Rα binding molecule, the PEG is a 40kD branched PEG comprising two 20kD arms.
The present invention also contemplates IL27 ra binding molecules comprising more than one PEG moiety, wherein the PEG has different size values, and thus a plurality of different PEGs are present at a particular ratio. For example, in the preparation of pegylated IL27 ra binding molecules, some compositions comprise a mixture of mono-, di-, tri-, and tetra-pegylated sdAb conjugates. In some compositions, the percentage of mono-PEGylated material is 18-25%, the percentage of di-PEGylated material is 50-66%, the percentage of trimerized ethylene glycol material is 12-16%, and the percentage of tetra-PEGylated material is as much as 5%. Such composite compositions may be produced by reaction conditions and purification methods known in the art. Chromatography can be used to resolve fractions of the conjugate and then identify fractions containing the conjugate with, for example, the desired amount of PEG attached, purified from unmodified protein sequences and conjugates with other amounts of PEG attached.
PEGylation often occurs at the N-terminal α -amino group of the polypeptide, the ε amino group on the side chain of the lysine residue and the imidazole group on the side chain of the histidine residue. Since most recombinant polypeptides have a single alpha group and several epsilon and imidazole groups, depending on the chemical nature of the linker, many positional isomers can be produced.
Two widely used first generation activated monomethoxy PEGs (mPEG) 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 preferentially react with lysine residues to form a carbamate linkage, but are also known to react with histidine and tyrosine residues. The N-terminal single site of the polypeptide is targeted by reductive amination using a PEG-aldehyde linker.
PEG can be bound to the IL27 ra binding molecules of the invention via a terminal reactive group ("spacer") that mediates binding between the free amino or carboxyl groups of one or more polypeptide sequences and polyethylene glycol. PEG with a spacer that can bind to a free amino group includes N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate of polyethylene glycol with N-hydroxysuccinimide.
In some embodiments, pegylation of sdabs is facilitated by incorporating unnatural amino acids with unique side chains to facilitate pegylation of specific sites. Incorporation of unnatural amino acids into polypeptides to provide functional moieties to achieve site-specific pegylation of such polypeptides is known in the art. See, e.g., ptacin, et Al, (PCT international application No. PCT/US2018/045257, month 8, day 3 of 2018 submitted and disclosed in month 2, day 7 of 2019, international publication No. WO 2019/028419Al.
The PEG moiety of the pegylated IL27 ra binding molecule may be linear or branched. Branched PEG derivatives, "star-PEG" and multi-arm PEG are contemplated by the present invention. Practice of the inventionUseful embodiment PEGs include 10kDa linear PEG-aldehydes (e.g.,ME-100AL, NOF U.S. Co (NOF America Corporation), north Bailao (One North Broadway), white Plains, N.Y. 10601 USA), 10kDa straight chain PEG-NHS ester (e.g.>ME-100CS,/>ME-100AS,ME-100GS,/>ME-100HS, NOF), 20kDa straight-chain PEG-aldehyde (e.g. +.>ME-200AL, NOF, 20kDa straight-chain PEG-NHS ester (e.g., +.>ME-200CS,/>ME-200AS,ME-200GS,/>ME-200HS, NOF), 20kDa 2-arm branched PEG-aldehyde, the 20kDa PEG-aldehyde comprising two 10kDa straight-chain PEG molecules (e.g., ->GL2-200AL3, NOF), 20kDa 2-arm branched PEG-NHS ester, 20kDa PEG-NHS ester, which Comprises two 10kDA linear PEG molecules (e.g., +.>GL2-200TS,/>GL200GS2, NOF), 40kDa 2-arm branched PEG-aldehyde, the 40kDa PEG-aldehyde comprising two 20kDa linear PEG molecules (e.g., ->GL2-400AL 3), 40kDa 2-arm branched PEG-NHS ester, the 40kDa PEG-NHS ester comprising two 20kDa linear PEG molecules (e.g., +.>GL2-400AL3,/>GL2-400GS2, NOF), straight-chain 30kDa PEG-aldehyde (e.g., +.>ME-300 AL) and straight-chain 30kDa PEG-NHS ester.
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 a "knob-to-hole" modification. Pestle and socket modifications are more fully described in Ridgway, et al (1996) protein engineering (Protein Engineering) 9 (7): 617-621 and U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,642,228, U.S. Pat. No. 7,695,936, and U.S. Pat. No. 8,216,805, both issued on 3 and 24, 5, 1, 2010, and 10, 2012. The knob-to-socket modification refers to modification of the interface between two immunoglobulin heavy chains in the CH3 domain, wherein: i) In the CH3 domain of the first heavy chain, the amino acid residues are replaced with amino acid residues having a larger side chain (e.g., tyrosine or tryptophan), creating a protuberance ("knob") from the surface and ii) in the CH3 domain of the second heavy chain, the amino acid residues are replaced with amino acid residues having a smaller side chain (e.g., alanine or threonine), creating a cavity ("socket") within the interface of the second CH3 domain, wherein the protruding side chain ("knob") of the first CH3 domain is received by the cavity in the second CH3 domain. In one embodiment, the "knob-to-socket modification" includes amino acid substitutions T366W and optionally S354C in one of the antibody heavy chains, and amino acid substitutions T366S, L368A, Y V and optionally Y349C in the other antibody heavy chain. Furthermore, the Fc domain can be modified by introducing a cysteine residue at position S354 on one chain and Y349 on the second chain, thereby creating a stable disulfide bond between the two antibody heavy chains of the Fc region (Carter, et al (2001) Immunol Methods 248,7-15). The knob-to-hole format is used to facilitate expression of a first polypeptide (e.g., sdAb that binds IL27 ra) on a first Fc monomer with a "knob" modification and a second polypeptide on a second Fc monomer with a "knob" modification to facilitate expression of a heterodimeric polypeptide conjugate.
Targeting domain
In some embodiments, the IL27 ra binding molecules are provided as components of multivalent (e.g., bivalent) fusion proteins having polypeptide sequences ("targeting domains") to facilitate selective binding to cell types or tissues that express cell surface molecules that specifically bind to such targeting domains, optionally with the inclusion of a linker molecule between the sdAb sequence that binds IL27 ra and the targeting domain sequence of the fusion protein.
In some embodiments of the IL27 ra binding molecules, the IL27 ra binding molecules can target cells of a particular cell type by incorporating a targeting domain into the structure of the IL27 ra 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 may be any moiety that specifically binds to one or more cell surface molecules (e.g., T cell receptors) expressed on the surface of a target cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is an il27rα+ T cell.
In some embodiments, the targeting domain is a ligand for a receptor. In some embodiments, the targeting domain is a ligand for a receptor expressed on the surface of a T cell. In some embodiments, the ligand is a cytokine. In some embodiments, cytokines include, but are not limited to, the group consisting of interleukins, interferons, and functional derivatives thereof. In some embodiments, cytokines include, but are not limited to, the following group: IL2, IL3, IL4, IL7, IL9, IL12, IL15, IL18, IL21, IL22, IL23, IL27, IL28, IL34 and modified forms or fragments thereof bind to their cognate ligands expressed on the surface of T cells. In some embodiments, cytokines include, but are not limited to, the following group: interferon alpha, interferon a2b, interferon gamma or interferon lambda and modified forms or fragments thereof, which bind to their cognate ligands expressed on the surface of T cells.
In another aspect, the present invention provides multivalent binding molecules comprising: (a) An IL27 ra binding molecule and (b) a second binding molecule that specifically binds to an extracellular domain of a second cell surface molecule, wherein the IL27 ra binding molecule and the second binding molecule are optionally operably linked by a chemical or polypeptide linker. In some embodiments, the IL27 ra binding molecules of the invention can be used to prepare multivalent binding molecules described in Gonzalez et al, PCT/US2018/021301 published as WO 2018/182935 A1, 10/4. In some aspects, the second binding molecule specifically binds to the following extracellular domain: (i) A component of a cytokine receptor that activates 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 the group consisting of: EGFR, erbB2, erbB3, erbB4, insR, IGF1R, insRR, PDGFR alpha, PDGFRbeta, 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 a TNFR superfamily member selected from the group consisting of: TNFR1 (TNFRSF 1A) TNFR2 (TNFRSF 1B; TNFRSF 2), 41-BB (TNFRSF 9); AITR (TNFRSF 18); BCMA (TNFRSF 17), CD27 (TNFRSF 7), CD30 (TNFRSF 8), CD40 (TNFRSF 5), death receptor 1 (TNFRSF 10C), death receptor-3 (TNFRSF 25), death receptor 4 (TNFRSF 10A), death receptor 5 (TNFRSF 10B), death receptor-6 (TNFRSF 21), decoy receptor-3 (TNFRSF 6B), decoy receptor 2 (TNFRSF 10D), EDAR, fas (TNFRSF 6), HVEM (TNFRSF 14), and pharmaceutical compositions containing the same,LTBR(TNFRSF 3), OX40 (TNFRSF 4), RANK (TNFRSF 11A), TACI (TNFRSF 13B), troy (TNFRSF 19), xedr (TNFRSF 27), osteoprotegerin (TNFRSF 11B), TWEAK receptor (TNFRSF 12A), BAFF receptor (TNFRSF 13C), NGF receptor (TNFRSF 16).
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 for a tumor cell receptor) selected from the group consisting of: GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Ra2, CD19, mesothelin, her2, epCam, muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, pan-ErbB and FAP.
In some embodiments, the targeting domain of the IL27 ra binding molecule is an antibody (as defined above, including, e.g., VHH, scFv, etc.). Examples of antibodies that can be incorporated as targeting domains for IL27 ra 0 binding molecules include, but are not limited to, the following groups: anti-GD 2 antibodies, anti-BCMA antibodies, anti-CD 19 antibodies, anti-CD 33 antibodies, anti-CD 38 antibodies, anti-CD 70 antibodies, anti-GD 2 antibodies and IL3Ra2 antibodies, anti-CD 19 antibodies, anti-mesothelin antibodies, anti-Her 2 antibodies, anti-EpCam antibodies, anti-Muc 1 antibodies, anti-ROR 1 antibodies, anti-CD 133 antibodies, anti-CEA antibodies, anti-PSMA antibodies, anti-EGRFRVIII antibodies, anti-PSCA antibodies, anti-GPC 3 antibodies, anti-Pan-ErbB antibodies and anti-FAP antibodies.
The antibody or antigen binding fragment thereof may also be linked to another antibody to form, for example, a bispecific or multispecific antibody
Markers
In some embodiments, the IL27 ra binding molecules of the invention are operably linked to a label. In some embodiments, the label is incorporated for use as an imaging agent, diagnostic agent, or for use in a cell sorting procedure. The term label includes, but is not limited to, fluorescenceA label, a bioactive 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 sdAb-binding IL27 ra (e.g., VHH-binding IL27 ra) molecule is stably associated (e.g., covalent, coordinated covalent) with the imaging marker. The term imaging marker is used to describe any of a variety of compounds characterized by an ability to facilitate the identification, tracking, and/or localization of sdabs (or metabolites thereof) that bind IL27 ra using diagnostic procedures. Examples of imaging markers include, but are not limited to, fluorescent compounds, radioactive compounds, and compounds that are opaque to the imaging method (e.g., X-rays, ultrasound). Examples of radioactive compounds used as imaging markers include, but are not limited to: technetium-99 m% 99m Tc), indium-111% 111 In, iodine-131% 131 I) Iodine-123% 123 I) Iodine-125% 125 I) Gallium-67% 67 Ga) and lutetium-177% 177 Lu and P 32 P, C 14 C) The tritium is 3 H) Yttrium [ ] 90 Y), actinium 225 Ac), astatine 211 At, re% 186 Re, bi% 212 Bi or Bi 213 Bi) and rhodium% 188 Rh)。
Therapeutic agent
In some embodiments, the IL27 ra binding molecules of the invention are operatively linked to a therapeutic agent. Examples of therapeutic agents include therapeutic small molecules (e.g., chemotherapeutic agents) or biologic therapeutic agents, including antibodies, cytotoxic or cytostatic compounds, radioisotopes, molecules of plant, fungal or bacterial origin, or biological proteins (e.g., protein toxins) or particles (e.g., nanoparticles or recombinant viral particles, e.g., by viral coating proteins), therapeutic antibodies, chemotherapeutic agents, as more fully described herein.
In some embodiments, the therapeutic agent that can incorporate an IL27 ra binding molecule of the invention is a short range radiation emitter, including, for example, a short range high energy a emitter. Examples of such radioisotopes include alpha emitters, beta emitters, gamma emitters or beta/gamma 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, bi-213% 213 Bi) and rhodium-188% 188 Rh)。
In some embodiments, the IL27Rα binding molecules of the invention are operably linked to a cytotoxic agent (or derivative thereof), such as maytansinol (maytansinol) or DM1 maytansine, a taxane or calicheamicin (calicheamicin), a pseudomonas exotoxin A, de-triangle Mei Danbai (deBouganin), ricin, diphtheria toxin, amatoxins, such as alpha-amanitin, saponin, maytansinoids, maytansine, auristatin, anthracyclines, calicheamicin, irinotecan, SN-38, du Kamei (duocarmycin), pyrrolobenzodiazepine(pyrrolobenzodiazepine), pyrrolobenzodiazepine>Dimer, indolobenzodiazepine +.>And indolobenzodiazepine +.>Dimer, or variant thereof).
Synthesis of IL27Rα binding molecules:
in some embodiments, the IL27 ra binding molecules of the invention are polypeptides. However, in some embodiments, only a portion of the IL27 ra binding molecule is a polypeptide, e.g., wherein the IL27 ra binding molecule comprises a non-peptidyl domain (e.g., a PEG IL27 ra binding sdAb conjugate, a radionucleotide IL27 ra binding sdAb conjugate, or a small molecule IL27 ra binding sdAb conjugate). The following provides guidance for solid phase and recombinant synthesis of the polypeptide portion (domain) of the IL27Rα binding molecules of the invention. In those embodiments in which only the portion of the IL27 ra binding molecule is a polypeptide, it is understood that one or more of the peptide-based domains of the IL27 ra binding molecule is an intermediate in the process, which may undergo further processing to complete synthesis of the desired IL27 ra binding molecule. The polypeptide domains of the IL27 ra binding molecules of the invention can be produced by conventional methods for constructing polypeptides, including recombinant or solid-phase synthesis, as described in more detail below.
Chemical synthesis
In addition to producing mutant polypeptides by expression of nucleic acid molecules that have been altered by recombinant molecular biotechnology, the polypeptide domain of the IL27 ra binding molecule can be chemically synthesized. Chemically synthesized polypeptides may be routinely produced by those skilled in the art. Chemical synthesis includes direct synthesis of peptides by chemical means of the polypeptide domain of the IL27 ra binding molecule that exhibits the described properties. The method may incorporate natural and unnatural amino acids at desired positions that facilitate the attachment of specific molecules (e.g., PEG).
In some embodiments, the polypeptide domain of the IL27 ra binding molecules of the invention can be prepared by chemical synthesis. Chemical synthesis of the polypeptide domain of the IL27 ra binding molecule can be performed by liquid or solid phase. Solid Phase Peptide Synthesis (SPPS) allows for the incorporation of unnatural amino acids and/or peptide/protein backbone modifications. Various forms of SPPS useful in synthesizing the polypeptide domains of the IL27Rα binding molecules of the present invention are known in the art (e.g., ganesan A. (2006) Mini Rev. Med. Chem.6:3-10; and Camarero et al, (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, the alpha functional group and any reactive side chains may be protected with acid labile or base labile groups, which are stable under conditions for amide linkage, but which can be readily cleaved without damaging the peptide chain that has been formed.
In solid phase synthesis, the N-terminal or C-terminal amino acid may be coupled to a suitable support material. Suitable support materials are those which are inert to the reagents and reaction conditions of the stepwise condensation and cleavage reactions in the synthesis process and which are insoluble in the reaction medium being used. Examples of commercially available support materials include styrene/divinylbenzene copolymers modified with reactive groups and/or polyethylene glycol; chloromethylated styrene/divinylbenzene copolymers; methylolated or aminomethylated styrene/divinylbenzene copolymers, and the like. The sequential coupling of protected amino acids can be performed according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.
At the end of the solid phase synthesis, the peptide is cleaved from the support material, simultaneously with cleavage of the side chain protecting group. The peptides obtained may be purified by a variety of chromatographic methods including, but not limited to, hydrophobic adsorption chromatography, ion exchange chromatography, partition chromatography, high Pressure Liquid Chromatography (HPLC) and reverse phase HPLC.
Recombinant production
Alternatively, the polypeptide domain of the IL27Rα binding molecules of the invention is produced by recombinant DNA techniques. In typical practice of recombinant production of a polypeptide, a nucleic acid sequence encoding a desired polypeptide is incorporated into an expression vector suitable for use in a host cell in which expression is to be accomplished, the nucleic acid sequence being operably linked to one or more expression control sequences encoded by the vector and functioning in the host cell of interest. If secretion leader sequences (signal peptides) are incorporated into the polypeptide, the recombinant protein may be recovered by disrupting the host cell or from the cell medium. The recombinant protein may be purified and concentrated for further use, including inclusion.
Synthesis of nucleic acid sequences encoding IL27Rα binding molecules
In some embodiments, the polypeptide domain of the IL27 ra binding molecule is produced by recombinant methods using a nucleic acid sequence encoding the polypeptide domain of the IL27 ra binding molecule (or a fusion protein comprising the polypeptide domain of the IL27 ra binding molecule). The nucleic acid sequence encoding the polypeptide domain of the desired IL27Rα binding molecule can be chemically synthesized using an oligonucleotide synthesizer.
The nucleic acid sequence is not limited to a sequence encoding a polypeptide; part or all of the non-coding sequence may also be included upstream or downstream of the coding sequence (e.g., the coding sequence of the polypeptide domain of an IL27 ra binding molecule). Routine procedures for isolating nucleic acid molecules are well known to those skilled in the art of molecular biology. For example, they may be generated by treating genomic DNA with a restriction endonuclease or by performing a Polymerase Chain Reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule may be produced, for example, by in vitro transcription.
The nucleic acid molecule encoding the polypeptide domain of the IL27 ra binding molecule (and fusions thereof) may comprise a naturally occurring sequence or a sequence that differs from a naturally occurring sequence, but which encodes the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules may consist of: RNA or DNA (e.g., genomic DNA, cDNA or synthetic DNA, e.g., produced by phosphoramidite-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, the nucleic acid molecule may be double-stranded or single-stranded (i.e., sense strand or antisense strand).
Nucleic acid sequences encoding the polypeptide domain of the IL27 ra binding molecule can be obtained from a variety of commercial sources that provide for the synthesis of custom nucleic acid sequences. Amino acid sequence variants of the HUMAN IL27 ra binding molecules of the invention are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code well known in the art. Such variants represent insertions, substitutions and/or specific deletions of the indicated residues. Any combination of insertions, substitutions and/or specific deletions may result in the final construct, provided that the final construct possesses the desired biological activity as defined herein.
Methods of constructing DNA sequences encoding the polypeptide domains of IL27 ra binding molecules and expressing these sequences in a suitable transformed host include, but are not limited to, the use of PCR-assisted mutagenesis techniques. Mutations consisting of amino acid residue deletions or additions may also be made into the polypeptide domain of the IL27 ra binding molecule using standard recombinant techniques. If deleted or added, the nucleic acid molecule encoding the polypeptide domain of the IL27Rα binding molecule is optionally digested with a suitable restriction endonuclease. The resulting fragments may be expressed directly or may be further manipulated by, for example, ligating to a second fragment. Ligation may be facilitated if the two ends of the nucleic acid molecule comprise complementary nucleotides that overlap one another, but blunt end fragments may also be ligated. PCR-generated nucleic acids can also be used to generate a variety of mutant sequences.
The polypeptide domains of the IL27Rα binding molecules of the invention can be produced not only recombinantly directly, but also in the form of 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. In general, the signal sequence may be a component of the vector or may be part of a 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 a signal sequence naturally associated with an IL27 ra binding molecule (i.e., a human IL27 ra signal sequence). The inclusion of the signal sequence depends on whether it is desired to secrete the IL27 ra binding molecule from the recombinant cell from which it is made. If the cell of choice is a prokaryotic cell, it is generally preferred that the DNA sequence does not encode a signal sequence. If the cell of choice is a eukaryotic cell, it is generally preferred to encode a signal sequence, most preferably a wild-type IL-2 signal sequence is used. Alternatively, heterologous mammalian signal sequences are also suitable, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretion leader sequences, such as the herpes simplex gD signal. When the recombinant host cell is a yeast cell, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), the alpha mating factor secretion signal sequence (alpha mating factor secretion signal sequence) may be used to effect extracellular secretion of the IL27 Ralpha binding molecule into the culture medium, as described in Singh, U.S. Pat. No. 7,198,919B1.
If the polypeptide domain of the IL27Rα binding molecule to be expressed is to be expressed in chimeric (chimera) form (e.g., a fusion protein comprising the 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 part of the polypeptide domain of the IL27Rα binding molecule and a second sequence encoding all or part of the heterologous polypeptide. For example, the polypeptide domain of the IL27 ra binding molecules described herein can be fused to a hexa-histidine tag to facilitate purification of a protein expressed by bacteria, or to a hexa-histidine, hemagglutinin, or Fc tag to facilitate purification of a protein expressed in eukaryotic cells. By first and second, it should not be understood that limiting the orientation of the elements of the fusion protein, the heterologous polypeptide may be linked to the N-terminus and/or C-terminus of the polypeptide domain of the IL27 ra binding molecule. For example, the N-terminus can be linked to a targeting domain and the C-terminus linked to a hexa-histidine tag purification handle.
The complete amino acid sequence of the polypeptide domain (or fusion/chimera) of the IL27Rα binding molecule to be expressed can be used to construct a back-translated gene. DNA oligomers containing a nucleotide sequence encoding a polypeptide domain of an IL27 ra binding molecule can be synthesized. For example, small oligonucleotides encoding part of the desired polypeptide may be synthesized and then ligated. Individual oligonucleotides typically contain 5 'or 3' overhangs for complementary assembly.
In some embodiments, the nucleic acid sequence encoding the polypeptide domain of the IL27 ra binding molecule may be "codon optimized" to facilitate expression in a particular host cell type. Techniques for codon optimization in a variety of expression systems, including mammalian, yeast, and bacterial host cells are well known in the art and there are online tools to provide codon optimized sequences for expression in a variety of host cell types. See, e.g., hawash, et al (2017) 9:46-53 and Mauro and Chappell inRecombinant protein expression in mammalian cells: methods and protocols (Recombinant Protein) Expression in Mammalian Cells: Methods and Protocols)，David Hacker (Human Press) new york. In addition, there are a variety of online network-based software that can be used for free to assist in preparing codon-optimized nucleic acid sequences.
Expression vector
After assembly (by synthesis, site-directed mutagenesis or another means), the nucleic acid sequence encoding the polypeptide domain of the IL27 ra binding molecule will be inserted into an expression vector. A variety of expression vectors for use in a variety of host cells may be used, typically based on the host cell selection 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, integrative vectors, and the like. Plasmids are examples of non-viral vectors. To facilitate efficient expression of recombinant polypeptides, the nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory sequences that function in the selected expression host.
Expression vectors typically contain a selection gene, also known as a selectable marker. The gene encodes a protein necessary for survival or growth of the transformed host cell grown in selective media. Host cells not transformed with vectors containing the selection gene cannot survive in the medium. Typical selection genes encode proteins that will (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline; (b) compensating for auxotroph-type defects; or (c) providing key nutrients not available from the complex media.
Expression vectors for the polypeptide domain of the IL27 ra binding molecules of the invention comprise regulatory sequences that are recognized by the host organism and are operably linked to the nucleic acid sequence encoding the polypeptide domain of the IL27 ra binding molecule. The terms "regulatory 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, e.g., goeddel (1990) in gene expression techniques: methods of enzymology (Gene Expression Technology: methods in Enzymology) 185 (Academic Press), san Diego, calif., U.S.A., regulatory sequences include nucleotide sequences that direct constitutive expression in many types of host cells, and nucleotide sequences that direct expression only in certain host cells (e.g., tissue-specific regulatory sequences). As will be appreciated by those skilled in the art, the design of expression vectors may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc., in the choice of expression control sequences, those factors will be considered as understood by those skilled in the art including, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject IL27Rα binding molecule, particularly with respect to possible secondary structures.
In some embodiments, the regulatory sequence is a promoter, the choice of which is based on, for example, the cell type in which expression is sought. A promoter is an untranslated sequence (typically within about 100 to 1000 bp) located upstream (5') of the start codon of a structural gene that controls the transcription and translation of a particular nucleic acid sequence to which it is operably linked. Such promoters generally fall into two categories, inducible and constitutive. Inducible promoters are promoters that increase transcription levels in response to certain changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature) from the initiation of DNA under their control. Numerous promoters recognized by a variety of potential host cells are well known.
The T7 promoter may be used in bacteria, the polyhedrin promoter may be used in insect cells, and the cytomegalovirus or metallothionein (metallothionein) promoter may be used in mammalian cells. In addition, tissue-specific and cell-specific promoters are widely used in the case of higher eukaryotic cells. These promoters are named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type in vivo. The skilled artisan is aware of the many promoters and other regulatory elements that can be used to direct expression of nucleic acids.
Transcription of the vector in the mammalian host cell may be controlled by, for example, promoters obtained from the viral genome, such as polyomavirus, vaccinia virus, adenovirus (e.g., human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (e.g., murine stem cell virus), hepatitis b virus, and most preferably simian virus 40 (SV 40), promoters from heterologous mammals, such as actin promoters, PGK (phosphoglycerate kinase), or immunoglobulin promoters, from heat shock promoters, so long as these promoters are compatible with the host cell system. The early and late promoters of SV40 virus are conveniently obtained as SV 40-restricted fragments which also contain the SV40 viral origin of replication.
Transcription in higher eukaryotes is typically increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, that act on a promoter to increase its transcription. Enhancers are relatively independent of orientation and position, found both 5 'and 3' to the transcriptional unit, within introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha fetoprotein and insulin). However, typically one will use enhancers from eukaryotic cell viruses. Examples include the SV40 enhancer on the posterior side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the replication origin, and adenovirus enhancers. Enhancers may be spliced into the expression vector 5' or 3' to the coding sequence, but are preferably located 5' from the promoter. Expression vectors for eukaryotic host cells will also contain the sequences necessary for termination of transcription and for stabilization of mRNA. Such sequences are generally obtainable from 5', occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. Construction of suitable vectors containing one or more of the above components employs standard techniques.
In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, the vector may contain an origin of replication and other genes encoding selectable markers. For example, the neomycin-resistance (neoR) gene confers resistance to G418 on its expressing cells, thus allowing phenotypic selection of transfected cells. Other examples of markers or reporter genes include beta-lactamase, chloramphenicol Acetyl Transferase (CAT), adenosine Deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine Kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyl transferase (XGPRT). One skilled in the art can readily determine whether a given regulatory element or selectable marker is suitable or unsuitable for use in a particular expression environment. Proper assembly of the expression vector can be confirmed by nucleic acid sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host.
Host cells
The invention further provides a prokaryotic or eukaryotic cell comprising and expressing a nucleic acid molecule encoding a polypeptide domain of an IL27 ra binding molecule. The cells of the invention are transfected cells, i.e. cells into which a nucleic acid molecule has entered, e.g. a nucleic acid molecule that has been introduced by recombinant DNA technology into a polypeptide domain encoding an IL27 ra binding molecule. Progeny of such cells are also considered to be within the scope of the invention.
Host cells are generally selected for their compatibility with the chosen expression vector, toxicity of the product encoded by the present IL27Rα binding molecule DNA sequence, their secretory properties, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and ease of purification of the product encoded by the DNA sequence. Suitable host cells for cloning or expressing the DNA in the vectors herein are the above-described prokaryotic cells, yeast or higher eukaryotic cells.
In some embodiments, recombinant IL27R alpha binding molecules of the polypeptide domain or its biologically active variants can also in eukaryotic cells in preparation, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (e.g., baculovirus vectors useful for protein expression in cultured insect cells (e.g., sf9 cells) include pAc series (Smith et al (1983) mol. Cell biol.3: 2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170: 31-39)); yeast cells (e.g., vectors for expression in yeast Saccharomyces cerevisiae (S. Cerenvisiae) (including 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 (Enje Corp. Invitrogen Corporation), santa Clago, california) and pPicZ (Enje Corp., santa Clago, california)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al (1987) EMBO J.6:187:195)).
Examples of useful mammalian host cell lines are mouse L cells (L-M [ TK- ], ATCC No. CRL-2648), monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (HEK 293 or HEK293 cell subclones grown in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR (CHO), mouse Sertoli cells (TM 4), monkey kidney cells (CV 1 ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1 587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo murine hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumors (T060562,ATCC CCL51), TRI cells, MRC 5 cells, MMF 4 cells, and human hepatoma cell lines (Hep G2). In mammalian cells, the control functions of expression vectors are typically provided by viral regulatory elements, e.g., promoters derived from polyoma, adenovirus type 2, cytomegalovirus and simian virus 40.
The polypeptide domain of the IL27 ra binding molecule can be produced in a prokaryotic host, such as bacterial e.coli, or in a eukaryotic host, such as an insect cell (e.g. Sf21 cells) or a mammalian cell (e.g. COS cells, NIH 3T3 cells or HeLa cells). These cells can be obtained from a number of sources, including the American type culture Collection (Ma Sana S, virginia). One skilled in the art can make this determination. In addition, if guidance is needed in selecting expression systems, those skilled in the art can review Ausubel et al (New Programming molecular biology protocols (Current Protocols in Molecular Biology), john Wiley and Sons, new York, 1993) and Pouwels et al (Cloning vector: laboratory Manual (A Laboratory Manual), 1985 journal 1987).
In some embodiments, the polypeptide domain of the recombinant IL27 ra binding molecule will be glycosylated or non-glycosylated depending on the host organism used to produce the IL27 ra binding molecule. If a bacterium is selected as the host, the polypeptide domain of an sdAb that binds IL27 ra may comprise a glycosylation motif, in particular an N-linked glycosylation motif of the sequence Asn-X-Ser (N-X-S) or Asn-X-Thr (N-X-T), where X is any amino acid other than proline. In such cases, it is desirable to eliminate such N-linked glycosylation motifs by modifying the sequence of the N-linked glycosylation motif to prevent glycosylation. In some embodiments, elimination of an Asn-X-Ser (N-X-S) N-linked glycosylation motif can be achieved by incorporating conservative amino acid substitutions at the Asn (N) residue and/or Ser (S) residue of the Asn-X-Ser (N-X-S) N-linked glycosylation motif. In some embodiments, elimination of an Asn-X-Thr (N-X-T) N-linked glycosylation motif can be achieved by incorporating conservative amino acid substitutions at Asn (N) residues and/or Thr (T) residues of the Asn-X-Thr (N-X-T) N-linked glycosylation motif. In some embodiments, elimination of As prokaryotic host cells does not provide a mechanism for recombinant protein glycosylation, and modification of the sequence to eliminate N-linked glycosylation sites can be avoided when a prokaryotic expression system is used to generate a recombinant cross-recombinant (crissrecombiant) sdAb that binds IL27Rα.
For other expression systems for prokaryotic and eukaryotic cells, see Sambrook et al (1989) molecular cloning: chapter 16 and 17 of the laboratory Manual (Molecular Cloning: A Laboratory Manual) (2 nd edition, cold spring harbor laboratory Press, prain, N.Y.). See, goeddel (1990) in gene expression technology: methods of enzymology (Gene Expression Technology: methods in Enzymology) 185 (academic Press, san Diego, calif.).
Transfection
The expression constructs can be introduced into a host cell to thereby produce the polypeptide domain of the recombinant IL27 ra binding molecules disclosed herein, or to produce biologically active muteins thereof. Vector DNA may be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al (1989) molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual) (2 nd edition, cold spring harbor laboratory Press, prain, N.Y.) and other standard molecular biology laboratory manuals.
To facilitate transfection of the target cells, the target cells may be directly exposed to the non-viral vector under conditions conducive to uptake of the non-viral vector. Examples of conditions that promote the uptake of exogenous nucleic acids by mammalian cells are well known in the art and include, but are not limited to, chemical means (e.g. Race-fern technology (Thermo-Fisher Scientific)), high salt and magnetic fields (electroporation).
Cell culture
The cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Mammalian host cells can be cultured in a variety of media. Commercially available media such as hami F10 (Ham's F) (Sigma), minimal essential media ((MEM), sigma), RPMI 1640 (Sigma) and darwinia modified i medium ((DMEM), sigma) are suitable for culturing host cells. Any of these media may be supplemented 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 equivalent energy sources, as desired. Any other necessary supplements may also be included in suitable concentrations known to those skilled in the art. The culture conditions, such as temperature, pH, etc., are those previously used to select the host cell for expression and will be apparent to one of ordinary skill.
Recombinant protein recovery
If a secretion leader is used, the recombinantly produced IL27Rα -binding polypeptide may be recovered from the culture medium as a secreted polypeptide. Alternatively, the IL27Rα binding polypeptides may be recovered from host cell lysates. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), may be used during the recovery stage from cell lysates to inhibit protease degradation during purification and antibiotics may be included to prevent the growth of exogenous contaminants.
Purification
Various purification steps are known in the art and can be sought, for example, by affinity chromatography. Affinity chromatography exploits the highly specific binding sites typically found in biological macromolecules to separate molecules according to their ability to bind to specific ligands. Covalent bonds attach the ligand to an insoluble porous support medium, allowing the ligand to be visibly present on the protein sample, thereby separating and purifying the second species from the mixture using the natural specific binding of one molecular species. Antibodies are commonly used for affinity chromatography. Size selection steps, such as gel filtration chromatography (also known as size exclusion or molecular sieve chromatography) may also be used for separation according to the size of the protein. In gel filtration, the protein solution is passed through a column packed with a semipermeable porous resin. The semipermeable resin has a pore size range that determines the size of the protein that can be separated using the column.
The polypeptide domain of the recombinant IL27 ra binding molecule produced by the transformed host may be purified according to any suitable method. The IL27Rα binding molecules can be isolated from inclusion bodies produced in E.coli or from conditioned medium of mammalian or yeast culture in which the particular IL27Rα binding molecule is produced using cation exchange, gel filtration, and or reverse phase liquid chromatography.
The substantially purified form of the recombinant polypeptide may be used, for example, as described herein, as a therapeutic agent.
The biological activity of the polypeptide domain of a recombinant IL27 ra binding molecule produced according to the above may be confirmed by IL27 ra binding using procedures known in the art, including but not limited to competition ELISA, radioligand binding assays (e.g., saturated binding, scatchard plot (Scatchard plot), non-linear curve fitting procedures, and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (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), using instruments commercially available from GE healthcare life Sciences (GE Healthcare Bio-Sciences), such as Biacore 8+, biacore S200, biacore T200 (GE Healthcare Bio-Sciences,100Results Way,Marlborough MA 01752)), liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR) and immunoprecipitation), and solid ligand binding assays (e.g., multi-well plate assays, bead-on ligand binding assays, on-column ligand binding assays and filtration assays).
Application method
Inhibition of IL27Rα Activity
In one embodiment, the invention provides methods of modulating the activity of an IL27Rα -expressing cell by administering to a subject an amount of an IL27Rα binding molecule sufficient to interfere with the activity of a receptor comprising IL27Rα. The invention also provides a method of modulating the activity of a cell expressing IL27 ra in a mixed population of cells, comprising contacting the population of cells with an IL27 ra binding molecule or complex of the invention in vivo and/or ex vivo in an amount sufficient to interfere with the activity of a receptor comprising IL27 ra.
Identification, isolation, enrichment or depletion of IL27Rα+ cells
In one embodiment, the invention provides methods of using the IL27rα binding molecules of the invention, which can be used in a process of isolating, enriching or depleting IL27rα+ cells from a biological sample comprising IL27rα+ cells. The biological sample may comprise blood-derived cells, such as PBMCs, cell culture-derived or tissue-derived (brain or bone marrow) B cells, T cells. Suitable processes for isolation, enrichment, or depletion of IL27Rα+ cells include centrifugation, filtration, magnetic cell sorting, and fluorescent cell sorting, using techniques well known in the art. The invention also provides a method of treating a subject suffering from a disease, disorder or condition by administering a therapeutically effective amount of a cell product enriched in or depleted of IL27rα+ cells using the IL27rα binding molecules described herein.
In one embodiment, the sorting procedure employs an IL27rα binding molecule comprising a fluorescent label for FACS separation or depletion of IL27rα+ cells from the sample. The fluorescent label can be attached to the sdAb of the IL27 ra binding molecule directly (e.g., optionally by chemical coupling using a linker) or indirectly (e.g., by biotinylation of the sdAb and binding of the biotinylated antibody to the streptavidin fluorescent dye conjugate). Such fluorescently labeled IL27rα+ cells can be isolated from the mixed cell population using conventional FACS techniques.
In an alternative embodiment, the selection procedure employs an IL27 ra binding molecule of the invention (e.g., a VHH that binds IL27 ra) that is coupled to a magnetic particle, providing a magnetic label of IL27 ra+ cells for use in a magnetic cell separation procedure. In one embodiment, the method comprises: (a) Coupling one or more IL27 ra binding molecules of the invention (e.g., IL27 ra-binding VHHs) to a magnetic particle; (b) Generating a mixture by contacting a biological sample with an amount of magnetic particles coupled to IL27 ra binding molecules; (c) Subjecting the magnetic field such that magnetically labeled il27rα+ cells are retained; (d) removing the non-magnetically labeled cells from the mixture; and (e) removing the magnetic field capable of isolating the IL27rα+ cells.
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 routine procedures to identify specific IL27rα+ or IL27rα -cell subsets that may be useful in research, diagnostic or clinical applications. For example, isolating a specific IL27rα+ T cell subset that also expresses one or more of CD4, CD8, CD19, CD25, and CD62L, magnetic field isolation by FACS or by techniques well known in the art, further iterates using one or more antibodies that specifically bind to CD4, CD8, CD19, CD25, and CD62L antigens, respectively.
In one embodiment, wherein the IL27 RA binding molecules can be used to deplete IL27 RA expressing cells from a biological sample comprising IL27 RA expressing cells, such peripheral blood or myeloid tissue, which can optionally be further treated to isolate human IL27 ra+ naive T cells from pre-selected cd4+ or cd8+ cells, or further isolate a subpopulation of IL27 ra+ naive T cells, or isolate human IL27 ra+ memory T cells from a population of cd4+ or cd8+ cells by depleting IL27 ra+ cells. In one embodiment, the IL27 ra binding molecules provide a method of producing a population of cells enriched for naive tregs from a biological sample, the method comprising depleting IL27 ra+ cells, optionally further comprising the step of depleting cd8+ and/or cd19+ cells using the IL27 ra binding molecules of the invention as described above. For a particular cell type, the IL27rα+ depleted cell population can optionally be further expanded in vitro to produce a cell product comprising a therapeutically effective amount of the IL27rα+ depleted cell product, which can be administered to a subject suffering from a disease, disorder, or condition. The population of IL27rα+ enriched cells can optionally be further expanded in vitro to produce a cell product comprising a therapeutically effective amount of IL27rα+ cells. Kit for detecting a substance in a sample
The invention also contemplates kits comprising pharmaceutical compositions of IL27 ra binding molecules. The kit is generally in the form of a physical structure containing the various components, as described below, and may be used, for example, to carry out the methods described above. The kit of the present invention may be designed to properly maintain the necessary conditions (e.g., refrigeration or freezing) of the components contained therein. The kit may further comprise a label or package insert, including identification information and instructions for use of the components therein. The components of the kit may be packaged in separate containers, and all of the various containers may be in a single package. The label or insert may include manufacturer information such as lot number and expiration date. The label or package insert may, for example, be integrated into the physical structure containing the components, contained separately in the physical structure, or affixed to the components of the kit (e.g., ampoule, syringe, or vial). The label or insert may be provided in physical form or on a computer readable medium. In some embodiments, the actual instructions are not present in the kit, but rather the kit provides a means of obtaining instructions from a remote location, for example, through an internet website, including by providing secure access to a password (or scannable code, such as a bar code or QR code on an IL27 ra binding molecule or container containing the kit) that complies with government regulations (e.g., HIPAA).
Examples
The following examples are presented to fully disclose and describe how to make and use the present IL27 ra binding molecules to those of ordinary skill in the art and are not intended to limit the scope of IL27 ra binding molecules that the inventors consider nor represent that they consider the experiments performed below to be all experiments performed. It should be understood that the exemplary descriptions are not necessarily written at the present time, but may be made to generate data or the like as described herein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Variations of the specifically described procedure employed may be obvious to one skilled in the art and it is expected that one skilled in the art may suitably employ such variations. Accordingly, the IL27 ra binding molecules are intended to be implemented in a manner different from that specifically described herein, and the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.
Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees celsius (°c), and pressure is at or near atmospheric pressure. Standard abbreviations are used, including the following: bp = base pair; kb = kilobase; pl = picoliter; s or sec = seconds; min = min; h or hr=hour; aa = amino acid; kb = kilobase; nt = nucleotide; pg = picogram; ng = nanogram; μg = micrograms; mg = milligrams; g = gram; kg = kg; dL or dL = deciliter; mu L or mu L = microliter; mL or mL = milliliter; l or l=l; μΜ=micromolar; mM = millimole; m = moles; kDa = kilodaltons; i.m. =intramuscular (intramuscular); p. = intraperitoneal (intraperitoneal); SC or SQ = subcutaneous (subcutaneous); QD = once daily; BID = twice daily; QW = once weekly; qm=once a month; HPLC = high performance liquid chromatography; BW = body weight; u = unit; ns = no statistical significance; PBS = phosphate buffered saline; PCR = polymerase chain reaction; nhs=n-hydroxysuccinimide; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = dado modified eagle medium; GC = genome copy; EDTA = ethylenediamine tetraacetic acid; PBMC = primary peripheral blood mononuclear cells; FBS = fetal bovine serum; FCS = fetal bovine serum; hepes=4- (2-hydroxyethyl) -1 piperazine ethanesulfonic acid; lps=lipopolysaccharide; ATCC = american type culture collection.
EXAMPLE 1 immunization protocol
Isolation of anti-hIL 27RαVHH was initiated by immunization of the camel with a polypeptide corresponding to amino acids 33-516 of hIL27Rα (UNIProt reference Q6UWB 1). The process of isolating anti-mIL 27RαVHH was initiated by immunization of a camel with the 201 amino acid extracellular domain of mIL27Rα, amino acids 25-510 of the precursor mIL27Rα (UNIPRT reference O70394). For each antigen, VHH were identified and isolated using the following method.
The synthetic DNA sequence encoding the antigen was inserted into pfuse_higg1_fc2 vector (merits biotechnology (Generay Biotechnology)) and transfected into HEK293F mammalian cell host cells for expression. The antigen was expressed as an Fc fusion protein, which was purified using protein a chromatography. The antigen was diluted with 1 XPBS (total antigen of about 1 mg). The quality was assessed by SDS-PAGE to ensure sufficient purity (> 80%) for immunization. The camel was adapted in the facility for at least 7 days prior to immunization. Immunization with antigen was performed, with antigen given once a week over a period of 7 weeks. For primary immunization, the immunogen was prepared as follows: 10mL of Complete Freund's Adjuvant (CFA) was added to the mortar, and then 10mL of 1 XPBS of the antigen was slowly added to the mortar with pestle and sample grinding until the antigen emulsified to milky and difficult to disperse. For the next six immunizations (2-7 weeks) in the immunization protocol, immunogens were prepared as described above, except that Incomplete Freund's Adjuvant (IFA) was used instead of CFA. About 2ml of emulsified antigen was subcutaneously injected on at least six sites on the camel, for a total of about 10ml per camel. When injecting antigen, the needle is kept in the subcutaneous space for about 10 to 15 seconds after each injection to avoid leakage of the emulsion.
EXAMPLE 2 phage library construction
Blood samples were collected from camels three days after the last injection in the immunization protocol. RNA was extracted from blood and transcribed into cDNA. The approximately 900bp reverse transcribed sequence encoding the VH-CH 1-hinge-CH 2-CH3 construct was isolated from the approximately the desired 700bp fragment encoding the VHH-hinge-CH 2-CH3 species. The purified about 700bp fragment was amplified by nested PCR. The amplified sequences were digested with Pst1 and Not 1. An approximately 400bp PST1/Not1 digestion fragment was inserted into the Pst1/Not1 digested pMECS phagemid vector such that the sequence encoding VHH is in frame with the DNA sequence encoding HA/His sequence. The PCR-generated sequences and vector of pMECS phagemid were digested with Pst I and Not I and subsequently ligated into pMECS/Nb recombinants. After ligation, the product was transformed into E.coli (E.coli) TG1 cells by electroporation. Transformants were enriched in growth medium and then transferred onto 2YT+2% glucose agar plates.
Example 3: isolation of antigen-specific VHHs
Phage libraries were biopanning to identify VHH that bound IL27 ra. 96-well plates were coated with IL27 ra and phage libraries were incubated in each well to allow phage expressing IL27 ra-reactive VHH to bind to IL27 ra on the plate. Washing the non-specifically bound phage and isolating the specifically bound phage. After selection, an enriched phage library expressing IL27 ra-reactive VHH was amplified in TG1 cells. The biopanning process described above was repeated 2-3 rounds to enrich the VHH-selective library for IL27 ra.
Example 4: identification of antibodies exhibiting specific binding to IFNgR 1:
after the biopanning of example 3 was completed, three single phage clones of 96-well plates were isolated for periplasmic extract ELISA (PE-ELISA) on IL27 ra coated plates to identify positive VHH binders that selectively bind IFNgR 1. 96-well plates were coated with IL27Rα and PBS under the same conditions. Next, the cells were closed at 37℃for 1 hour. Then, 100. Mu.l of the extracted antibody was added to each well and incubated for 1 hour. Subsequently, 100. Mu.l of anti-tag polyclonal antibody conjugated to HRP was added to each well and incubated at 37℃for 1 hour. Plates were developed with TMB substrate. The reaction was stopped by adding H2SO 4. The absorbance at 450nm was read on a microtiter plate reader. Antibodies with antigen coated wells that have at least three times higher absorbance than PBS coated controls are believed to exhibit specific binding to IL27 ra. Positive clones were sequenced and the sequences were analyzed to identify unique clonotypes
Example 5 evaluation of binding affinity by surface plasmon resonance
Representative examples were selected from each of the hIL27RαVHH clonotypes generated according to examples 1-3 for use in performing binding assessment by SPR as follows. Binding affinity of hIL27Rα binding molecules corresponding to SEQ ID NOS 2-27 was evaluated using Surface Plasmon Resonance (SPR) essentially following the following procedure. All experiments were performed on a Biacore T200 instrument equipped with a protein A derived sensor chip (Cytiva) in 10mM Hepes, 150mM NaCl, 0.05% (v/v) polysorbate 20 (PS 20) and 3mM EDTA (HBS-EP+ buffer). The mono-Fc VHH ligand flowed at a rate of 5. Mu.l/min for a variable time of 18 to 300 seconds to achieve the capture loadings listed in the following table. After ligand capture, IL27Rα -receptor extracellular injection is performed in high performance or single cycle kinetic mode A 2-fold dilution series of domains, the sequence modified to comprise a C-terminal poly-His sequence, typically comprising at least 5 concentrations between 1 μm and 1 nM. Surface regeneration was achieved by flowing 10mM glycine-HCl, pH 1.5 (60 seconds, 50. Mu.L/min). The sensorgrams minus the buffer were processed with Biacore T200 evaluation software and globally fitted with a 1:1langmuir binding model (global offset set to zero) to extract the kinetic and affinity constants (k) a ，k d ，k D )。R Maximum value <100RU indicates that the surface density is compatible with kinetic analysis. The calculated R is generated using the following formula Maximum value Value: r is R Maximum value Valence x of the load (RU) x ligand (molecular weight of analyte/molecular weight of ligand). Surface Activity is defined as R for experiment/calculation Maximum value Ratio of the two components. The results of these binding affinity experiments are provided in table 6.
It is to be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, serial numbers, patents and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Claims (13)

1. An IL27 ra binding molecule that specifically binds to the extracellular domain of IL2 Rb.
2. The IL27 ra binding molecule of claim 1, wherein the IL2Rb binding molecule comprises a single domain antibody (sdAb).
3. The IL27 ra binding molecule of claim 2, wherein the sdAb comprises complementarity determining regions 1 (CDR 1), CDR2, and CDR3, as shown in the rows of the following table:
4. the IL27 ra binding molecule of claim 2 or 3, wherein the sdAb has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity or 100% identity to any polypeptide sequence selected from SEQ ID NOs 2-25.
5. The IL27 ra binding molecule of claim 2, wherein the sdAb comprises complementarity determining regions 1 (CDR 1), CDR2, and CDR3, as shown in the rows of the following table:
6. the IL27 ra binding molecule of claim 2, wherein the sdAb has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity or 100% identity to any polypeptide sequence selected from the group consisting of SEQ ID NOs 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, and 174.
7. The IL27 ra binding molecule of any one of claims 3 or 5, wherein the sdAb is humanized or comprises CDRs grafted to a heterologous framework region.
8. The IL27 ra binding molecule of any one of claims 1-7, further comprising a labeling agent, imaging agent, and/or therapeutic agent.
9. The IL27 ra binding molecule of any one of claims 1-8, for isolating, depleting, or enriching IL27 ra+ cells of a biological sample.
10. A nucleic acid sequence encoding the IL27 ra binding molecule of any one of claims 1-8.
11. A recombinant viral or non-viral vector comprising the nucleic acid of claim 11.
12. A host cell comprising the nucleic acid of claim 11.
13. A kit comprising the IL2Rb binding molecule of any one of claims 1-8.
CN202180068078.5A 2020-08-05 2021-08-04 IL27Rα binding molecules and methods of use Pending CN116723859A (en)

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