Antibodies targeting interleukin-36R, their preparation methods, and applications

Abstract

This application provides antibodies targeting interleukin-36R, as well as their preparation and applications. Specifically, this application provides IL-36R antibodies that bind to IL-36R with high affinity, inhibit the binding of IL-36R ligands (α, β, γ) to IL-36R, and inhibit the signaling pathway activated by the IL-36R ligands, thereby treating and / or preventing IL-36-related diseases with high affinity and high biological activity.

Description

[Technical Field]

[0001] This application relates to the field of biopharmaceuticals, specifically to antibodies targeting interleukin-36R, their preparation methods, and applications.

[0002] Conventional technology The IL-1 receptor family (IL1R) includes at least 11 members, including IL1RI (IL1R1), IL1RII (IL1R2), IL1RAcP (IL1R3), ST2 (T1 / IL1R4), IL18Ra (IL1Rrp / IL1R5), IL1Rrp2 (IL1RL2 / IL1R6 / IL-36R), IL18Rb (AcPL / IL1R7), IL1RAPL1 (TIGIRR2 / IL1R8), and TIGIRR1 (IL1R9). IL-36R recognizes three different activating ligand cytokines—L36α, IL36β, and IL36γ (also known as IL-1F6, IL-1F8, and IL-1F9)—and can trigger the expression of inflammatory cytokines. Furthermore, it possesses two natural antagonists, IL-36Ra (IL-1F5) and IL-38, which, when bound to IL-36R, suppress the expression of inflammatory cytokines.

[0003] IL-36R is expressed in lung epithelial cells, cerebral vascular cells, kidney cells, testes, monocytes, skin-derived keratinocytes, fibroblasts, and endothelial cells. IL-36R consists of an extracellular ligand-binding domain, a transmembrane helix, and an intracellular signaling Toll / IL-1 receptor domain (TIR domain). IL-36α, IL-36β, and IL-36γ first bind to IL-36R, forming a signaling complex with IL-1RAcP. This then recruits myeloid differentiation factor 88 (MyD88), activating the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways mediated by c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases (ERK1 / 2). This process generates numerous inflammatory mediators, mediating inflammatory responses and playing a crucial role in adaptive immunity. The binding of IL-36Ra to IL-36R does not lead to the recruitment of IL-1RAcP, thus preventing the initiation of a pro-inflammatory cascade and enabling the anti-inflammatory properties of IL-36Ra. In a healthy human body, the signal activation effects mediated by IL-36α, IL-36β, and IL-36γ and the signal inhibition effects mediated by IL-36Ra are in balance. However, if a gene mutation occurs in IL-36Ra, the inhibitory function is inactivated, disrupting the balance and leading to inflammatory diseases.

[0004] The IL-36 cytokine is involved in certain severe forms of psoriasis, such as generalized pustular psoriasis (GPP) and palmoplantar pustulosis (PPP). GPP is a severe, lethal form of generalized pustular psoriasis. PPP is a chronic form of pustular psoriasis affecting the palms of the hands and soles of the feet. Currently, treatments for GPP and PPP include oral retinoids and topical steroids, but these have poor efficacy and severe side effects.

[0005] Therefore, in this technological field, there is a need for anti-IL-36R antibodies that bind to IL-36R with high affinity, inhibit the binding of IL-36α, IL-36β, and IL-36γ to IL-36R, and treat IL-36-related diseases such as GPP and PPP. [Overview of the project]

[0006] The object of this application is to provide an antibody targeting interleukin-36R, a method for preparing the antibody, and its applications. Specifically, the object of this application is to provide an IL-36R antibody with high affinity and high biological activity that can bind to IL-36R with high affinity and inhibit the binding of IL-36α, IL-36β, IL-36γ to IL-36R, and its applications. Another objective of this application is to provide an interleukin-36R binding molecule and its uses, particularly in the treatment and / or prevention or diagnosis of IL-36-related diseases such as psoriasis.

[0007] In a first aspect of the present invention, the heavy chain variable region of an antibody is provided, which includes the following three complementarity-determining regions (CDRs): CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO: 5.

[0008] In some embodiments, any of the amino acid sequences in the amino acid sequence includes a derivative sequence in which at least one (one to two, or even just one, such as one to three) amino acids are added, deleted, modified, and / or substituted, thereby maintaining the binding affinity to IL-36R. In some embodiments, the heavy chain variable region includes a human FR region or a mouse FR region. In some embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:9.

[0009] A second aspect of the present application provides a heavy chain of an antibody having the heavy chain variable region described in the first aspect of the present application.

[0010] In some embodiments, the heavy chain of the antibody also includes a heavy chain constant region. In some embodiments, the heavy chain constant region is a human region, a mouse region, or a rabbit region.

[0011] A third aspect of the present invention provides an antibody light chain variable region comprising the following three complementarity-determining regions (CDRs): CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and CDR3' shown in SEQ ID NO: 8.

[0012] In some embodiments, any of the amino acid sequences in the amino acid sequence include at least one (one to two, or even just one, such as one to three) optionally selected amino acids added, deleted, modified, and / or replaced, a derivative sequence that maintains the binding affinity of IL-36R. In some embodiments, the light chain variable region includes a human FR region or a mouse FR region. In some embodiments, the light chain variable region has the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 10, 11, or 12.

[0013] A fourth aspect of the present application provides an antibody light chain having the light chain variable region described in the third aspect of the present application.

[0014] In some embodiments, the light chain of the antibody also includes a light chain constant region. In some embodiments, the light chain constant region is a human region, a mouse region, or a rabbit region.

[0015] In the fifth aspect of the present application, (1) the heavy chain variable region described in the first aspect of the present application, and / or (2) the light chain variable region described in the third aspect of the present application, or an antibody having the heavy chain described in the second aspect of the present application and / or the light chain described in the fourth aspect of the present application is provided.

[0016] In some embodiments, the dissociation constant KD (M) of the antibody binding to the human IL-36R protein (e.g., wild type) is (0.5 - 10) × 10 -11 and may be (1 - 6) × 10 -11 or may be (1 - 2) × 10 -11 . In some embodiments, the dissociation constant KD (M) of the antibody binding to human IL-1 Rrp2 is (0.5 - 10) × 10 -11 and may be (1 - 6) × 10 -11 or may be (1 - 2) × 10 -11 . In some embodiments, the antibody can inhibit the binding of IL-36α, IL-36β, IL-36γ to IL-36R. In some embodiments, the antibody can inhibit cytokine secretion mediated by IL-36α, IL-36β, IL-36γ, and the cytokines include IL-6, IL-8, and GM-CSF. In some embodiments, the antibody is selected from animal antibodies, chimeric antibodies, humanized antibodies, or combinations thereof. In some embodiments, the antibody is a double-stranded antibody or a single-stranded antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a partially or fully humanized monoclonal antibody. In some embodiments, the heavy chain variable region sequence of the antibody is as shown in SEQ ID NO:1 or 9, and / or the light chain variable region sequence of the antibody is as shown in SEQ ID NO:2, 10, 11 or 12. In some embodiments, the heavy chain variable region sequence of the antibody is as shown in SEQ ID NO:1, and the light chain variable region sequence of the antibody is as shown in SEQ ID NO:2. In some embodiments, the heavy chain variable region sequence of the antibody is as shown in SEQ ID NO:9, and the light chain variable region sequence of the antibody is as shown in SEQ ID NO:10, 11 or 12. In some embodiments, the antibody is in the form of a drug conjugate.

[0017] In the sixth aspect of the present application, (i) the heavy chain variable region described in the first aspect of the present application, the heavy chain described in the second aspect of the present application, the light chain variable region described in the third aspect of the present application, the light chain described in the fourth aspect of the present application, or the antibody described in the fifth aspect of the present application, and (ii) a recombinant protein comprising an optionally selected tag sequence that aids in expression and / or purification is provided.

[0018] In some embodiments, the tag sequence includes a 6His tag. In some embodiments, the recombinant protein (or polypeptide) described above includes a fusion protein. In some embodiments, the recombinant protein is a monomer, dimer or polymer.

[0019] In the seventh aspect of the present application, the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to IL-36R, and a CAR construct is provided in which the scFv has the heavy chain variable region described in the first aspect of the present application and the light chain variable region described in the third aspect of the present application.

[0020] The eighth aspect of the present application provides recombinant immune cells expressing the exogenous CAR construct described in the seventh aspect of the present application.

[0021] In some embodiments, the immune cells are selected from a group consisting of NK cells and T cells. In some embodiments, the immune cells are derived from humans or non-human mammals (e.g., mice).

[0022] In the ninth aspect of this application, (a) The antibody portion is selected from the group consisting of the heavy chain variable region described in the first aspect of the present application, the heavy chain described in the second aspect of the present application, the light chain variable region described in the third aspect of the present application, the light chain described in the fourth aspect of the present application, or the antibody described in the fifth aspect of the present application or a combination thereof, and (b) The antibody-drug conjugate is provided in which the binding portion that binds to the antibody portion is selected from the group consisting of a detectable marker, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.

[0023] In some embodiments, the antibody portion and the binding portion are linked by a chemical bond or a linker.

[0024] In the tenth aspect of the present application, an active ingredient is provided, the active ingredient being selected from the group consisting of a heavy chain variable region as described in the first aspect of the present application, a heavy chain as described in the second aspect of the present application, a light chain variable region as described in the third aspect of the present application, a light chain as described in the fourth aspect of the present application, or an antibody as described in the fifth aspect of the present application, a recombinant protein as described in the sixth aspect of the present application, an immune cell as described in the eighth aspect of the present application, an antibody-drug conjugate as described in the ninth aspect of the present application, or a combination thereof, the active ingredient being used in (a) a measurement reagent or kit, and / or (b) the preparation of a pharmaceutical for the prevention and / or treatment of IL-36-related disease.

[0025] In some embodiments, the IL-36-related disease is an inflammatory disease mediated by IL-36. In some embodiments, the IL-36-related disease is a disease caused by overstimulation or mutation of the IL-36 cytokine. In some embodiments, the IL-36-related disease is selected from the group consisting of inflammation, autoimmune diseases, or a combination thereof, and may be, for example, an autoimmune disease. In some embodiments, the IL-36-related disease is selected from the group consisting of psoriasis, scleroderma, chronic kidney disease, inflammatory bowel disease, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory arthritis, asthma, allergies, or a combination thereof, and may be, for example, psoriasis. In some embodiments, the psoriasis includes psoriasis vulgaris, erythrodermic psoriasis, psoriatic arthritis, generalized pustular psoriasis (GPP), and palmoplantar pustulosis (PPP). In some embodiments, the drug is used to inhibit the binding of IL-36α, IL-36β, IL-36γ to IL-36R. In some embodiments, the drug is used to inhibit cytokine secretion mediated by IL-36α, IL-36β, and IL-36γ. In some embodiments, the antibody is in the form of a drug conjugate (ADC). In some embodiments, the measurement reagent or kit is used for the diagnosis of IL-36-related diseases. In some embodiments, the measurement reagent or kit is used to measure the IL-36R protein in a sample. In some embodiments, the measuring reagent is a measuring chip.

[0026] In the eleventh aspect of this application, (i) an active component selected from the group consisting of the heavy chain variable region described in the first aspect of this application, the heavy chain described in the second aspect of this application, the light chain variable region described in the third aspect of this application, the light chain described in the fourth aspect of this application, or the antibody described in the fifth aspect of this application, the recombinant protein described in the sixth aspect of this application, the immune cells described in the eighth aspect of this application, the antibody-drug conjugate described in the ninth aspect of this application, or a combination thereof, and (ii) To provide a pharmaceutical composition containing a pharmaceutically acceptable vector.

[0027] In some embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, the pharmaceutical composition is an injectable preparation.

[0028] In the twelfth aspect of the present application, the polynucleotide code is (1) A heavy chain variable region as described in the first aspect of this application, a heavy chain as described in the second aspect of this application, a light chain variable region as described in the third aspect of this application, a light chain as described in the fourth aspect of this application, or an antibody as described in the fifth aspect of this application, (2) Recombinant protein described in the sixth aspect of the present application, (3) Provides a polynucleotide selected from polypeptides of the group consisting of CAR constructs described in the seventh aspect of the present application.

[0029] A thirteenth aspect of the present application provides a vector containing the polynucleotide described in the twelfth aspect of the present application.

[0030] In some embodiments, the vector includes bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses and retroviruses, or other vectors.

[0031] A fourteenth aspect of the present application provides a genetically modified host cell containing the vector described in the thirteenth aspect of the present application, or having the polynucleotide described in the twelfth aspect of the present application incorporated into its genome.

[0032] In the fifteenth aspect of this application, (1) In vitro, the sample comes into contact with the antibody described in the fifth aspect of this application or the antibody-drug conjugate described in the ninth aspect of this application, (2) A method is provided for in vitro testing (including diagnostic or non-diagnostic) of IL-36R protein in a sample, comprising a procedure to measure whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of IL-36R protein in the sample.

[0033] In some embodiments, the method is non-diagnostic and non-therapeutic.

[0034] In the sixteenth aspect of this application, a measurement plate is provided which includes a plate (support plate) and a test piece containing the antibody described in the fifth aspect of this application or the antibody-drug conjugate described in the ninth aspect of this application.

[0035] In the seventeenth aspect of this application, (1) A first container containing the antibody described in the fifth aspect of this application, and / or (2) A second container containing a secondary antibody against the antibody described in the fifth aspect of this application, Alternatively, a kit including the measuring plate described in the sixteenth aspect of this application is provided.

[0036] In the eighteenth aspect of this application, (a) Culturing the host cells described in the fourteenth aspect of this application under conditions suitable for expression, (b) A method for preparing recombinant polypeptides is provided, comprising the step of isolating a recombinant polypeptide from a culture, the recombinant polypeptide being an antibody according to the fifth aspect of the present application or a recombinant protein according to the sixth aspect of the present application.

[0037] A nineteenth aspect of the present application provides a method for the prevention and / or treatment of IL-36-related disease, comprising administering to a subject of interest the antibody described in the fifth aspect of the present application, an antibody-drug conjugate of the antibody, CAR-T cells expressing the antibody, or a combination thereof.

[0038] In some embodiments, the method further includes administering combination therapy with other pharmaceuticals or treatments to the subjects in need.

[0039] Of course, within the scope of this application, it is understood that the above-mentioned technical features of this application and the technical features specifically described below (for example, in the examples) can be combined to form new technical solutions. Due to space limitations, a detailed explanation will not be provided here. [Brief explanation of the drawing]

[0040] The specific features of the invention described herein are as stated in the claims. The features and advantages of the invention described herein can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail herein. A general description of the accompanying drawings is as follows. [Figure 1] Biological activity measurement of humanized candidate antibodies - results of cytokine IL-6 release. [Figure 2] Biological activity measurement of humanized candidate antibodies - results of cytokine IL-8 release. [Figure 3] Biological activity measurement of humanized candidate antibodies - results of cytokine GM-CSF release. [Figure 4] Biological activity measurement of humanized candidate antibodies - NF-κB phosphorylation results. [Figure 5] Cell-binding activity of candidate antibodies to human IL-36R. [Figure 6] Cell-binding activity of candidate antibodies to monkey IL-36R. [Figure 7] Functional inhibitory effects of candidate antibodies against huIL-36 stimulants in NCI / ADR-RES cells. [Figure 8] Inhibitory effect of candidate antibodies against huIL-36 stimulating factors in HIF cells. [Figure 9] Inhibitory effect of candidate antibodies against huIL-36 stimulating factors in HIF cells. [Figure 10] Inhibitory effect of candidate antibodies against huIL-36 stimulating factors in HIF cells. [Figure 11] The effect of candidate antibodies on the clinical score (PASI) of imiquimod-induced psoriasis in cynomolgus monkeys. [Figure 12] Effect of candidate antibodies on HE staining pathological scores of imiquimod-induced psoriasis cynomolgus monkey skin tissue.

[0041] Specific Embodiments The embodiments of the present invention will be described below using specific examples, but those familiar with this art will be able to easily understand other advantages and effects of the present invention as disclosed herein.

[0042] Term definition As a result of extensive and thorough research, the applicant obtained an IL-36R antibody with surprisingly high affinity and high biological activity. Tests have shown that the IL-36R antibody of this application can treat and / or prevent IL-36-related diseases by binding to IL-36R with high affinity, inhibiting the binding of IL-36R ligands (α, β, γ) to IL-36R, and inhibiting the signaling pathway activated by IL-36R ligands. Based on this, the applicant has completed this application.

[0043] term As used herein, the terms “administration” and “treatment” mean the application of an exogenous drug, therapeutic agent, diagnostic agent or composition to an animal, human, subject, cell, tissue, organ or biological fluid. The terms “administration” and “processing” may also refer to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods and experimental methods. Processing of cells includes contact between reagents and cells, contact between reagents and liquids, and contact between liquids and cells. The terms “administration” and “processing” may also mean by the binding of reagents, diagnostic agents, or compositions, or by the processing of cells in vitro and independently. “Processing,” when applied to humans, animals or subjects, means therapeutic procedures, preventive procedures, research and diagnostics, and includes contact of IL-36R antibodies with humans or animals, subjects, cells, tissues, physiological compartments or physiological fluids.

[0044] As used herein, the term “treatment” means administering, either orally or topically, a therapeutic agent comprising the IL-36R antibody or any of its compositions to a patient having one or more symptoms of a disease for which the therapeutic agent is known to be therapeutic. The patient is usually administered a therapeutically effective dose of the therapeutic agent that is effective in alleviating one or more symptoms of the disease.

[0045] As used here, the terms "arbitrarily" or "optionally" mean that the event or situation described below may occur, but is not necessarily to occur. For example, "arbitrarily one to three antibody heavy chain variable regions are selected" means that antibody heavy chain variable regions with specific sequences may be present, but are not required, and there may be one, two, or three of them.

[0046] antibody As used here, the term "antibody" refers to an immunoglobulin, which has a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Because the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, their antigenicity also differs. This allows us to divide immunoglobulins into five classes, or different types of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with the heavy chain constant regions corresponding to the different immunoglobulins being α, δ, ε, γ, and μ. IgG represents the most important class of immunoglobulins and can be divided into four subclasses—IgG1, IgG2, IgG3, and IgG4—based on differences in chemical structure and biological function. The light chains are divided into κ or λ chains based on differences in the constant region. The subunit structures and three-dimensional arrangements of the different classes of immunoglobulins are well known to those skilled in the art.

[0047] The amino acid sequences of approximately 110 amino acids near the N-terminus of the antibody's heavy and light chains are significantly different and are therefore designated as the variable region (V region), while the amino acid sequence near the C-terminus is relatively stable and is therefore designated as the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively unvariable FR regions (FRs). The amino acid sequences of the four FRs are relatively unvariable and do not directly participate in the binding reaction. The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). The light chain variable region (LCVR) and the heavy chain variable region (HCVR) each consist of three CDR regions and four FR regions, arranged from the amino terminus to the carboxyl terminus as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain, i.e., the closed hypervariable regions (LCDRs), refer to LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain, i.e., the heavy chain hypervariable regions (HCDRs), refer to HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment described in this invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) or the Kabat and Chothia numbering rules (HCDR1). The four FR regions in the native heavy chain and light chain variable regions basically exhibit a β-sheet structure connected by three CDRs that form a connecting loop, and can optionally form a partial β-sheet structure. The CDRs of each chain are held in close proximity to each other via the FR regions and, together with the CDR of the other chain, form the antigen-binding site of the antibody. The amino acids constituting the FR or CDR regions can be identified by comparing the amino acid sequences of the same type of antibody. The constant region does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as being involved in antibody-dependent cytotoxicity.

[0048] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment that possesses antigen-binding activity. An Fv antibody contains the antibody heavy chain variable region and light chain variable region, but lacks a constant region, and contains the smallest antibody fragment with all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains, enabling it to form the structure necessary for antigen binding.

[0049] As used herein, the term “antigenic determinant” refers to a discontinuous three-dimensional site on an antigen recognized by the antibody or antigen-binding fragment of this invention.

[0050] This application includes not only complete antibodies, but also immunoactive antibody fragments or fusion proteins formed with antibodies and other sequences. Therefore, this application also includes the aforementioned antibody fragments, derivatives, and analogs.

[0051] In this application, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric or humanized monoclonal antibodies containing human and non-human portions, can be prepared by DNA recombination techniques well known in the art.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific and target a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone cell line.

[0053] As used here, the term "chimeric antibody" refers to an antibody molecule expressed by inserting a chimeric gene, created by splicing the V-region gene of a mouse antibody with the C-region gene of a human antibody, into a vector and transfecting it into host cells. This not only maintains the high specificity and affinity of the parent mouse antibody, but also allows its human Fc segment to effectively mediate biological effects.

[0054] As used herein, the term "humanized antibody" refers to a variant of the variable region of the mouse antibody of this application, which has a CDR region derived from (or substantially derived from) a non-human antibody (such as a mouse monoclonal antibody), an FR region substantially derived from a human antibody sequence, and a constant region. In other words, the CDR region sequence of a mouse antibody is transplanted into a different type of human germline antibody framework sequence. Since the CDR sequence is responsible for most of the antibody-antigen interaction, recombinant antibodies that mimic the characteristics of specific native antibodies can be expressed by constructing an expression vector.

[0055] In this application, the antibody may be monospecific, bispecific, triplicate, or have multiple specificities.

[0056] In this application, the antibody also includes its conserved variants, in which up to 10, 8, 5, and 3 amino acids, compared to the amino acid sequence of the antibody, are replaced with amino acids having similar or analogous properties to form polypeptides. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table A.

[0057] [Table A]

[0058] Anti-IL-36R antibody As used herein, the term "IL-36R" generally refers to natural or recombinant human IL-36R, and non-human homologs of human IL-36R. Unless otherwise specified, the molar concentration of IL-36R is calculated using the molecular weight of the IL-36R homodimer.

[0059] This application provides an antibody comprising a heavy chain and a light chain that exhibits high specificity and high affinity for IL-36R, wherein the heavy chain contains the amino acid sequence of the heavy chain variable region (VH), and the light chain contains the amino acid sequence of the light chain variable region (VL).

[0060] For example, the CDR of the heavy chain variable region (VH) is selected from the following group. CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:5, and / or The CDR of the light chain variable region (VL) is selected from the following group. CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and CDR3' shown in SEQ ID NO: 8.

[0061] Here, any of the amino acid sequences in the above amino acid sequence includes a derivative sequence in which at least one amino acid (which may be one to two, or even one, such as one to three) is added, deleted, modified, and / or substituted, thereby maintaining the binding affinity of IL-36R.

[0062] In some embodiments, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid may be an amino acid sequence having at least 80% homology, at least 85%, at least 90%, and at least 95%.

[0063] The antibody of this invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, or humanized antibodies, and may be a humanized antibody, a human-animal chimeric antibody, or a completely humanized antibody.

[0064] The antibody derivatives described in this application may include single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more antibodies of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes.

[0065] Here, the animal may be a mammal such as a mouse.

[0066] The antibody of this invention may be a mouse antibody, chimeric antibody, humanized antibody, CDR-implanted antibody, and / or modified antibody targeting human IL-36R.

[0067] In one embodiment, any one or more sequences in SEQ ID NO:3, 4, and 5, or sequences having L-36R binding affinity formed by adding, deleting, modifying, and / or substituting at least one amino acid, are located in the CDR region of the heavy chain variable region (VH).

[0068] In one embodiment, any one or more sequences in SEQ ID NO: 6, 7, and 8, or sequences having L-36R binding affinity formed by adding, deleting, modifying, and / or substituting at least one amino acid, are located in the CDR region of the light chain variable region (VL).

[0069] In one embodiment, VH CDR1, CDR2, and CDR3 are each independently selected from one or more sequences in SEQ ID NO:3, 4, and 5, or sequences having L-36R binding affinity formed by adding, deleting, modifying, and / or substituting at least one amino acid, and VL CDR1, CDR2, and CDR3 are each independently selected from one or more sequences in SEQ ID NO:6, 7, and 8, or sequences having L-36R binding affinity formed by adding, deleting, modifying, and / or substituting at least one amino acid.

[0070] In the foregoing description of this application, the number of added, deleted, modified and / or substituted amino acids may be, for example, 40% or less, 35% or less, 1-33%, 5-30%, 10-25%, or 15-20% of the total amount of amino acids in the original amino acid sequence.

[0071] In this application, the number of added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4, or 5, and may be 1-3, 1-2, or 1.

[0072] Antibody preparation Any method suitable for producing a monoclonal antibody may produce the anti-IL-36R antibody of this invention. For example, animals may be immunized with a conjugated or naturally occurring IL-36R homodimer or a fragment thereof. Appropriate immunization methods, such as adjuvants, immunostimulants, or booster immunizations, may be used, and one or more routes may be used.

[0073] Any suitable form of IL-36R can be used as an immunogen (antigen) to produce IL-36R-specific non-human antibodies, allowing for screening of the biological activity of such antibodies. The stimulating immunogen may be full-length mature human IL-36R containing a natural homodimer, or a peptide containing one or more epitopes. The immunogen may be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen may be naturally occurring and purified, or it may be produced from genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed using a suitable gene vector, which may include, but is not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, plasmids, and nonviral vectors.

[0074] An exemplary method for preparing the anti-human IL-36R antibody of this application is described in Example 1.

[0075] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody is an IgG antibody, and the IgG1 isotype is used. Optimization of the sequence of the required constant structural domain can be achieved by screening the antibody with the biological measurements described in the following examples to obtain the desired biological activity.

[0076] Here again, both classes of light chains can be used in the compounds and methods of this specification. Specifically, κ, λ chains or their variants are available in the compounds and methods of this application.

[0077] An exemplary method for humanizing the anti-human IL-36R antibody of this application is described in Example 2.

[0078] The DNA molecule sequences of the antibody or its fragments according to this invention can be obtained by conventional techniques such as amplification using PCR or genome library screening. Furthermore, single-chain antibodies can be formed by fusing the coding sequences of the light chain and heavy chain.

[0079] Once the desired sequence is obtained, it can be acquired in large quantities by recombination. This is usually done by cloning it into a vector, transplanting it into cells, and then isolating the desired sequence from the proliferated host cells using conventional methods.

[0080] Furthermore, especially when the fragment length is short, it is possible to synthesize the sequence artificially. In many cases, very long fragments can be obtained by synthesizing several smaller fragments and ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) or cells known in this art.

[0081] This application also relates to vectors comprising a suitable DNA sequence and a suitable promoter or regulatory sequence as described above. These vectors can be used to transform suitable host cells so that they can express proteins.

[0082] The host cell may be a prokaryotic cell such as a bacterial cell; a lower eukaryotic cell such as a yeast cell; or a higher eukaryotic cell such as a mammalian cell. Examples of animal cells include CHO-S, CHO-K1, and HEK-293 cells (however, it is not limited to these).

[0083] The steps described herein for transforming host cells with recombinant DNA can be carried out using techniques well known in the art. The resulting transformants can be cultured by conventional methods and express the polypeptide encoded by the gene of this application. Depending on the host cells used, they may be cultured under appropriate conditions using conventional culture media.

[0084] Typically, transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention. The antibody of the present invention can then be purified using conventional immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, and conventional separation and purification methods known to those skilled in the art.

[0085] The obtained monoclonal antibodies can be identified by conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Antibody-drug conjugates (ADCs) Furthermore, this application provides an antibody-drug conjugate (ADC) based on the antibody of this application.

[0086] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is bound to the chemically conjugable effector molecule. Here, the effector molecule may be a drug having therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0087] The antibody of this application and the effector molecule may be coupled via a coupling agent. The coupling agent may be one or more of the following: a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, or a coupling agent using a disulfide bond. The non-selective coupling agent refers to a compound that covalently bonds the effector molecule and the antibody, such as glutaraldehyde. The carboxyl group coupling agent may be one or more of the following: a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) or an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0088] Specific residues on antibodies (such as Cys and Lys) are used for binding to functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as glycol polymers), and therapeutic agents. Antibodies can form antibody-functional agent complexes by binding to functional agents. Functional agents (drugs, measurement reagents, stabilizers, etc.) are bound to antibodies (covalently). Functional agents can be bound to antibodies directly or indirectly via linkers.

[0089] Antibodies can form antibody-drug conjugates (ADCs) by binding to drugs. Typically, ADCs contain a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in the intracellular environment, for example, by degradation at the desired site, thereby releasing the drug from the antibody. Suitable degradable linkers include enzymatically degradable linkers, such as linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or carbohydrate linkers containing glucuronides that can be degraded by glucuronidases. Peptidyl group linkers may include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze when the pH is below a certain level, such as hydrazone) and linkers that degrade under reducing conditions (e.g., disulfide linkage linkers). Typically, non-degradable linkers release drugs under conditions where the antibody is hydrolyzed by a protease.

[0090] Before binding to the antibody, the linker has an activating group that can react with a specific amino acid residue, and binding is achieved via this activating group. Sulfhydryl-specific activating groups may include, for example, maleimide compounds, halogenated amides (e.g., iodized, brominated, or chlorinated), halogenated esters (e.g., iodized, brominated, or chlorinated), halogenated methyl ketones (e.g., iodized, brominated, or chlorinated), benzyl halides (e.g., iodized, brominated, or chlorinated), vinyl sulfones, pyridyl disulfide, mercury derivatives such as 3,6-di-(mercurymethyl)dioxane whose counterion is an acetate, chloride, or nitrate, and polymethylenedimethyl sulfide thiosulfonates. The linker may include, for example, maleimide that binds to the antibody via thiosuccinimide.

[0091] The drug may be any cytotoxic, cell proliferation inhibitory, or immunosuppressive drug. In some embodiments, the linker binds the antibody to the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug may have an amino group, carboxyl group, sulfhydryl group, hydroxyl group, or carbonyl group capable of forming a bond with the linker. In the case of a drug directly bound to the linker, the drug has a reactive group before binding to the antibody. Useful drug types include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, and vinca alkaloids. In this application, the drug-linker is used to form an ADC in a simple one-step process. In other embodiments, a bifunctional linker compound is used to form an ADC in a two-step or multi-step process. For example, in the first step, a cysteine ​​residue reacts with the reactive portion of the linker, and in subsequent steps, the functional group of the linker reacts with the drug to form an ADC.

[0092] Typically, functional groups on the linker are selected to facilitate specific reactions with appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety is used for specific reactions with reactive alkynyl groups on the drug moiety. The drug is covalently bonded to the linker via a 1,3-dipolar cycloaddition reaction between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides), isocyanates and isothiocyanates (suitable for reactions with amines and alcohols), and activated esters such as N-succinimidyl esters (suitable for reactions with amines and alcohols). These and other bonding strategies are well known to those skilled in the art, as described in *Studies in Biomaterials*, 2nd edition (Elsevier). Those skilled in the art will understand that when complementary reactive functional groups are selected for selective reactions between the drug moiety and the linker, each member of the complementary pair can be used in both the linker and the drug.

[0093] Furthermore, the present invention provides a method for preparing an antibody-drug conjugate (ADC), which may further include conjugating an antibody with a drug-linker compound under conditions sufficient to form an ADC.

[0094] In some embodiments, the method of the present invention includes conjugating an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker complex. In these embodiments, the method of the present invention further includes conjugating the antibody-linker complex to a drug moiety under conditions sufficient to covalently bond the drug moiety to the antibody via the linker.

[0095] In these embodiments, the antibody-drug conjugate (ADC) is represented by the following molecular formula. [ka] Here Ab stands for antibody, LU stands for linker, D is a drug The subscript 'p' is a value chosen from 1 to 8.

[0096] application This application provides uses for the antibody of this application, for example, for the preparation of diagnostic agents or for the preparation of drugs to prevent and / or treat IL-36-related diseases. The IL-36-related diseases include, but are not limited to, inflammatory diseases, autoimmune diseases, and inflammatory diseases including psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), osteoarthritis, rheumatoid arthritis (RA), rheumatoid arthritis or osteoporosis, inflammatory fibrosis (scleroderma, pulmonary fibrosis and sclerosis, etc.), asthma (including allergic asthma), hypersensitivity and cancer.

[0097] Pharmaceutical composition Furthermore, the present application provides compositions. In certain embodiments, the complex is a drug conjugate comprising the antibody or its activated fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable vector. Typically, these substances may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, in which case the pH may typically be about 5 to 8, for example, the pH may be about 6 to 8, and the pH may vary depending on the properties of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral administration, intraperitoneal administration, intravenous administration, or topical administration.

[0098] Furthermore, the antibody described in this application may be a cell therapy drug expressed intracellularly by a nucleotide sequence, such as the antibody for chimeric antigen receptor T cell immunotherapy (CAR-T). Since the pharmaceutical composition of this application can directly bind to the IL-36R protein molecule, it can be used for the prevention and treatment of IL-36R-related diseases. It can also be used in combination with other therapeutic agents.

[0099] The pharmaceutical composition of this application comprises the monoclonal antibody (or its complex) of this application, and a pharmaceutically acceptable vector or excipient, in a safe and effective amount (for example, 0.001-99 wt%, 0.01-90 wt%, or 0.1-80 wt%), and a pharmaceutically acceptable vector or excipient. Such vectors include, but are not limited to, sodium chloride, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The formulation needs to be adapted to the dosage form. The pharmaceutical composition of this application can be prepared, for example, by conventional methods from physiological saline or an aqueous solution containing glucose and other excipients, and made into an injectable preparation. Pharmaceutical compositions such as injectable preparations and solutions should preferably be prepared under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the polypeptide of this application can be used in combination with other therapeutic agents.

[0100] When using a drug composition, a safe and effective amount of the drug composition should be administered to the mammal, where a safe and effective amount is typically at least about 10 mg / kg body weight, and in most cases about 50 mg / kg body weight or less, and the dose may range from about 10 mg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage should take into account further factors such as the manner of administration and the patient's health condition, all of which are within the scope of the skills of a skilled physician.

[0101] Measurement applications and kits The antibody of this invention can be used, for example, in measurement applications to measure a sample and thereby provide diagnostic information.

[0102] In this application, the samples used include cell samples, tissue samples, and biopsy samples. In this application, the term "biopsy" includes all types of biopsies known to those skilled in the art. Therefore, the biopsies used in this application may consist, for example, of tissue samples prepared by endoscopic methods or by organ puncture or needle biopsy.

[0103] The samples used in this application include fixed or preserved cell or tissue samples.

[0104] The present invention further provides a kit comprising the antibody (or a fragment thereof), which may include, for example, a container, instructions for use, a buffer, and the like. For example, the antibody may be immobilized on a measurement plate.

[0105] The antibody or fragment relating to this invention can have the following excellent technical effects. (a) The antibody of this invention has excellent biological activity and specificity. (b) Compared to mouse antibodies and chimeric antibodies, the humanized antibody of this invention has comparable affinity to IL-36R while exhibiting lower immunogenicity. (c) Inhibition of IL-36R by the antibody of this invention can significantly inhibit the expression pathway of inflammatory factors in patients. (d) The antibody of this invention has an affinity for IL-36R of some non-human mammals (such as monkeys) that is equivalent to that of human IL-36R, and can be used for testing and quality control in animal models. (e) The antibody of this invention, by binding to IL-36R, blocks and inhibits the activation signaling pathway that binds IL-36α, IL-36β, and IL-36γ to IL-36R, thereby reducing inflammatory cytokines (IL-6, IL-8, GM-CSF, etc.).

[0106] The present application will be described in more detail below with reference to specific examples. It should be understood that these examples are used solely to illustrate the present application and not to limit its scope. In the following examples, experimental methods for which specific conditions are not given generally follow conventional conditions, such as those described in Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and quantities refer to weight percentages and weight quantities. [Examples]

[0107] Example 1 The method for producing mouse-derived monoclonal antibodies utilized the hybridoma production technique invented by Kohler and Milstein in 1975 (Nature, 1975, 256: 495-497). Human IL-36R Fc-tagged protein (ACRO,#IL2-H5254) and Freund's adjuvant were emulsified, and multiple strains of mice were immunized. After four immunizations, serum was collected, titer was measured by ELISA, and mice best suited for fusion of spleen cells and SP2 / 0 myeloma cells were selected. After screening hybridoma polyclonal cells with HAT, the binding activity of the polyclonal culture supernatant to human IL-36R, monkey IL-36R, and human IL1R1, as well as the inhibitory effect of the polyclonal antibodies on IL-36 stimulants, were measured. We selected the optimal polyclones, performed monocloning, similarly screened the binding and functional activity of the monoclonals, measured affinity using the Biacore method, and finally performed sequence analysis on the selected best hybridoma monoclonal cell lines.

[0108] [Table 1] As shown in Table 1, after screening a large number of hybridomas, the supernatant of the hybridoma(1)-B8,111(1)-E5 antibody showed high binding activity to human IL-36R and monkey IL-36R proteins at the cell binding level, but did not specifically bind to human IL1R1 protein. It also showed a clear functional blockade effect against cytokines stimulated by IL-36 stimulants (IL-36α, IL-36β, IL-36γ).

[0109] Example 2: Cloning and humanization of the variable region gene sequence of an anti-IL-36R antibody. 2.1 Cloning of antibody variable region genes in hybridoma cells The variable region cDNA sequences of mouse antibodies expressed by hybridoma cell lines were cloned based on the principles of TAKARA's 5'RACE technology. Briefly, heavy-chain and light-chain variable region gene-specific cDNAs were synthesized using the SMARTer 5'RACE synthesis kit (TAKARA, catalog number 634859) according to the instructions. The 5' and 3' ends of the cDNA sequences were modified with PCR primers. These primers are designed to add appropriate read sequences to the heavy-chain and light-chain variable region cDNAs, respectively, enabling seamless cloning of the resulting PCR products into existing recombinant antibody expression heavy-chain vectors pHB-Fc and light-chain vectors pHB-Cκ. The pHB-Fc expression vector contains the human IgG1 heavy-chain constant region gene sequence, where CH2 contains L234A and L235A (Eu numbering) mutations that weaken the ADCC effect of the antibody. The pHB-Cκ vector contains the human κ-expressing constant region gene sequence. The heavy and light chain variable region PCR amplification products were cloned into expression vectors using an in-fusion cloning reagent (TAKARA, catalog number 639650) to obtain human-mouse chimeric antibody expression vectors, which were then used to transform E. coli DH5α transformation-receptor cells (YB Biotech, catalog number FYE607-80VL). Monoclonal colonies were selected for Sanger sequencing and analyzed to obtain antibody variable region sequences.

[0110] As a result, an anti-IL-36R mating antibody (Huabo catalog number 900497) was obtained in which the light chain was derived from hybridoma 6(1)-B8 of Example 1 and the heavy chain was derived from hybridoma 111(1)-E5 of Example 1. Its variable region sequence is as follows. 900497 HCVR SEQ ID NO:1 QVQLQQTGSVLVRPGTSVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGEIHPNSAKTNYNEKFKGKATLTVDTFSSTAYVDLSSLASEDSAVYYCARVDYGKPWFAYWGQGTLVTVSA 900497 LCVR SEQ ID NO:2 QIVLTQSPTIMSASPGERVTMTCSASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTINSMEAEDAATYYCQQFQSSPLTFGAGTKLGLK Here, the underlined parts are CDRs (defined in IMGT, with the following arrays): [Table 2]

[0111] Expression of chimeric antibodies The expression vector obtained in 2.1 was amplified in E. coli, and plasmids sufficient for transient transfection of the chimeric antibody were prepared using an endotoxin removal plasmid extraction kit (TianJian Biotech, catalog number DP117). The host cells used for expression were CHO-S cells (Thermo Fisher, catalog number R80007). Two separately prepared heavy-chain and light-chain vectors were mixed with polyetherimide (PEI, Polyscience, catalog number 24765-1) to form liposome complexes, and CHO-S cells were transfected and cultured in a carbon dioxide shaker for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a protein A affinity chromatography column to obtain human-mouse chimeric antibodies.

[0112] 2.2 Humanization of mouse-derived anti-human IL-36R antibody - Method for preparing humanized antibodies Antibody humanization was performed using the following method: The variable region sequence of the chimeric antibody (Huabo catalog number 900497) was compared with available sequences in the NCBI IgBlast database, and through identification and analysis, human-derived framework regions (FR regions) suitable for constructing CDR-transplanted heavy and light chains were ultimately determined.

[0113] Modifications were designed according to the conserved and important amino acid residues in the FR region of the human antibody. Humanization mutations were designed for the variable regions of the heavy and light chains of the chimeric antibody, respectively, and humanization point mutation antibody expression plasmids were amplified and constructed using PCR technology. These humanization point mutation antibody expression plasmids were expressed in CHO-S cells and purified to obtain humanized antibody proteins. Indicators such as affinity and activated cytokine release of the humanized antibodies were screened using Biacore and cell biological activity measurements, and three humanized anti-IL-36R antibodies with superior performance were obtained. The numbers of the obtained humanized anti-IL-36R antibodies are 900513, 900527, and 900534, and their VH and VL sequences are shown below. 900513, 900527, and 900534 HCVR SEQ ID NO:9 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMHWAKQAPGQGLEWIGEIHPNSAKTNYNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARVDYGKPWFAYWGQGTLVTVSS 900513 LCVR SEQ ID NO:10 EIVLTQSPGTLSLSPGERATLSCSASSSVSSSYLHWYQQKPGQAPRLWIYSTSNRATGIPDRFSGSGSGTDFTLTISRLEPEDFATYYCQQFQSSPLTFGQGTKLEIK 900527 LCVR SEQ ID NO:11 QIVLTQSPGTLSLSPGERATLSCRASSSVSSSYLHWYQQKPGQAPRLWIYSTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFATYYCQQFQSSPLTFGQGTKLEIK 900534 LCVR SEQ ID NO:12 QIVLTQSPGTLSLSPGERATLSCRASSSVSSSYLHWYQQKPGQAPRLLIYSTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFATYYCQQFQSSPLTFGQGTKLEIK Here, the underlined parts are CDRs (defined in IMGT), and 900513, 900527, and 900534 are three humanized antibody protein numbers, respectively. The CDR regions of humanized antibodies 900513, 900527, and 900534 are the same as the CDR region of chimeric antibody 900497, and the FR region of the humanized antibodies is mutated based on chimeric antibody 900497.

[0114] Example 3: Measurement of the biological activity of humanized anti-human IL-36R antibody 3.1 Antibody affinity measurement using Biacore (SPR) The test materials and equipment are as follows: Human IL-1 Rrp2 / IL-1 R6 Protein with His Tag, ACRO Biosystems, IL2-H52H6 HBS-EP+(10X), GE, BR-1006-69 Series S Sensor Chip CM5,GE,29-1275-56 His Capture Kit, GE, 28995056 BIACORE, GE, Biacore 8K pH 1.5 Glycine solution, GE, BR100354

[0115] The testing method is as follows. The Series S Sensor Chip CM5 chip was equilibrated at room temperature for 20-30 minutes, and then the chip was placed in the instrument. Following the instructions for the His Capture Kit, the anti-His antibody was immobilized onto the Series S Sensor Chip CM5 chip. HBS-EP+(10X) solution was diluted 10-fold with ultrapure water to be used as the running buffer. The antigen (His-tagged human IL-1 Rrp2 / IL-1 R6 protein) was then added to the running buffer. The antigen was diluted to 1 μg / ml and injected at a rate of 10 μL / min for 15 seconds, capturing approximately 50 RU of antigen. The antibody samples were diluted with equilibrium buffer to concentrations of 30 nM, 15 nM, 7.5 nM, 3.75 nM, 1.875 nM, 0.9375 nM, and 0.46875 nM. The electrophoresis buffer was set to zero concentration, and the samples were bound at 30 μL / min for 120 s, dissociated for 900 s, and the tips were regenerated with a pH 1.5 glycine solution. The samples were analyzed using a capture method single-cycle kinetic program. After selecting the corresponding analysis program and analyzing the data, it was confirmed that there was no significant reference binding. Then, Kinetics was selected, and fitting analysis was performed using a 1:1 binding model to determine the kinetic parameters of the samples.

[0116] [Table 3] Note: 900389 is an anti-human IL-36R antibody developed by BI Corporation, and Huabo synthesized the variable region gene using the sequence described in the patent (WO2013 / 074569A1) and expressed it independently. Specifically, the heavy chain variable region of 900389 is 81B4vH33_90vH shown in SEQ ID NO:89 of WO2013 / 074569A1, and the light chain variable region of 900389 is 81B4vK32_105vK shown in SEQ ID NO:77 of WO2013 / 074569A1. Both 81B4vH33_90vH and 81B4vK32_105vK were obtained by humanization modification of the mouse antibody 81B4, and the constant region of 900389 is the same as the candidate antibody of this application.

[0117] The affinity constants (KD(M)) for each humanized antibody to human IL-1 Rrp2 are shown in Table 3. From these results, the affinity of the humanized monoclonal antibody of this application is 10 -11 This was close to the order of [the specified order], indicating extremely strong affinity.

[0118] 3.2 Measurement of the inhibitory effect of anti-human IL-36R antibodies on huIL-36 stimulating factors Add 100 μL per well of the human ovarian cancer cell line NCI / ADR-RES to a 96-well cell culture plate (4.5 x 10⁶ cells per well). 4 The samples were added using ) and incubated overnight at 37°C and 5% CO2, and then serially diluted. Subsequently, 50 μL / well was added and incubated at 37°C and 5% CO2 for 15 minutes, and then 50 μL / well of ligand huIL-36α (1 μg / mL), huIL-36β (80 ng / mL), and huIL-36γ (120 ng / mL) were added, mixed well, and incubated at 37°C and 5% CO2 for 18-24 hours. The cell culture supernatant was centrifuged and measured using the Biolegend ELISA kit: huIL-6 (1:100 dilution, catalog number 430503), huIL-8 (1:5 dilution, catalog number 431503), and huGM-CSF (1:2 dilution, catalog number 432003). Measurement of NF-κB activation effect in NCI / ADR-RES cells under huIL-36β stimulation: Ligand huIL-36β was diluted to 240 ng / mL and added to cells pre-incubated with the test antibody. The cells were incubated at 37°C, 5% CO2 for 30 minutes. The supernatant was removed by centrifugation, the cells were lysed, and the lysate was taken out. Phosphorylation of NF-κB was measured using a kit (Cisbio, catalog number 64NFBPEG).

[0119] [Table 4] As shown in Figures 1-4 and Table 4, the humanized antibodies 900513, 900527, and 900534 have a significant inhibitory effect on the function of the human stimulants IL-36α, IL-36β, and IL-36γ, and can significantly inhibit the secretion of cytokines IL-6, IL-8, and GM-CSF, as well as the phosphorylation of NF-κB.

[0120] Example 4 Nonspecific binding test (SPR) of chimeric antibody and humanized antibody This test measures the nonspecific adsorption effect of antibodies and non-target molecules using the SPR method. The equipment, instruments, and reagents used in the test are shown in Tables 5 and 6. [Table 5] [Table 6] Series S Sensor Chip CM5 (GE, #BR-1005-30) chips were equilibrated at room temperature for 20-30 minutes and then set in a Biacore 8K (GE) instrument. Egg-derived lysozyme solution (Sigma, #L3790) and soy-derived trypsin inhibitor type 1-S (Sigma, #T-2327) were immobilized onto the CM5 chips using an amino group coupling kit (GE, #BR-1000-50). HBS-EP (1X) (GE, #BR-1006-69) was used as the injection buffer, and four equilibration circles were set up. Polyclonal rabbit anti-lysozyme (ABcam, Ab391), anti-trypsin inhibitor antibody (LifeSpan Biosciences, #LS-C76609), chimeric antibody, and humanized antibody were each diluted to 1000 nM in equilibrium buffer. A flow rate of 5 μL / min was set to inject channels 1, 2, and 3, and Flow Cells 1 and 2. The binding time was 10 min and the dissociation time was 15 min. The regeneration flow rate was set to 50 μL / min, and regeneration was performed for 60 seconds with 0.85% phosphoric acid solution (ProteOn, 176-2260), followed by regeneration for 30 seconds with 50 mM sodium hydroxide solution. Here, sample 900389 is a humanized anti-human IL-36R antibody developed by BI, with Huabo synthesizing the variable region gene and expressing it independently based on a patent. Sample 900497 is an anti-IL-36R chimeric antibody constructed by Huabo's hybridoma screening.

[0121] As a result, as shown in Table 7, the binding signal between 900389 and the egg-derived lysozyme solution exceeded 20 RU, suggesting the presence of nonspecific electrostatic and hydrophobic binding. The binding signals between 900497 and the egg-derived lysozyme solution and the large-derived trypsin inhibitor 1-S type were all less than 20, suggesting the absence of nonspecific electrostatic and hydrophobic binding. [Table 7] Note: After subtracting the effect of the buffer, it is generally considered that if the response value is less than 20 RU, the interaction is weak and negligible; if it is greater than 20 RU, there is a clear interaction; and if it is greater than 100 RU, there is a strong interaction.

[0122] Example 5: Cell binding and functional testing of stable cell line antibodies Stable cell lines were constructed by inserting the BI control sequence 900389, the Huabo-screened humanized sequence 900527, and TNP IgG1 (Fc silence) 900543 into Huabo GS vectors, constructing recombinant expression plasmids, and transfecting each plasmid into CHO-K1 cells. The protein number of the stable cell line constructed with Huabo 900527 was HB0034, and the expression level of the screened monoclonal cell line exceeded 4 g / L after optimization of the upstream process. Antibodies expressed by the stably transfected cell lines were purified and measured by flow cytometry binding and functional assays. Sample 900543 is a TNP IgG1 (Fc silence) negative control antibody.

[0123] 5.1 Flow cytometry binding assay of stable cell line antibodies We conducted binding activity studies of stable cell line antibodies to human IL-36R and monkey IL-36R. The cell line used for the flow cytometry studies was the CHO-K1 cell line, constructed in Huabo, expressing human IL-36R and monkey IL-36R. The test results are shown in Table 8 and Figures 5 and 6. [Table 8] Huabo's monoclonal antibody HB0034 showed high binding activity to both human IL-36R and monkey IL-36R. Compared to the positive control antibody 900389, the present antibody had a lower EC50 value and higher binding activity to human IL-36R.

[0124] 5.2 Measurement of the inhibitory effect of stable cell lines on huIL-36 stimulating factor 5.2.1 Inhibitory effect of candidate antibodies on huIL-36 stimulating factors in NCI / ADR-RES cells The human ovarian cancer cell line NCI / ADR-RES was added to a 96-well cell culture plate at 100 μL per well (the number of cells per well was 4.5 x 10 4 ), incubated overnight at 37°C and 5% CO2, and the samples were serially diluted. Then, it was added at 50 μL / well, incubated at 37°C and 5% CO2 for 15 minutes, the ligand huIL-36β (40 ng / mL) was added at 50 μL / well, mixed well, and then cultured at 37°C and 5% CO2 for 18 - 24 hours. The cell culture supernatant was centrifuged, and huIL-6 was measured using the HTRF kit (Cat#62HIL06PEG, CISBIO). The results are as shown in Table 9 and Figure 7.

Table 9

[0125] 5.2.2 Functional inhibitory effect of candidate antibodies against huIL-36 stimulating factors in HIF cells HIF (Human Intestinal Fibroblasts) cells were prepared into a cell suspension of 4.5E5 / ml in Serum Starvd medium, 100 ul (45000 cells) per well was added to 96wp and incubated overnight. After the antibody was serially diluted, 50 ul / well was added and incubated at 37°C for 15 minutes. IL-36α was diluted to 1000 ng / ml, 50 ul per well was added, and mixed well. It was cultured at 37°C, 5% CO2 for 4 hours, the supernatant was recovered by centrifugation, and the content of human IL-6 was measured. IL-36β was diluted to 80 ng / m, 50 ul per well was added, and mixed well. It was cultured at 37°C, 5% CO2 for 4 hours, the supernatant was recovered by centrifugation, and the content of human IL-6 was measured. IL-36β was diluted to 40 ng / m, 50 ul per well was added, and mixed well. It was cultured at 37°C, 5% CO2 for 20 minutes, and the content of pNFκB was measured using the kit. The results are shown in Table 10 and Figure 8, Table 11 and Figure 9, and Table 12 and Figure 10. [Table 10] [Table 11] [Table 12] These results indicate that the stable cell line humanized antibody HB0034 exhibits a clear functional inhibitory effect on the human stimulants IL-36α and IL-36β, significantly inhibiting the secretion of the cytokine IL-6 and the phosphorylation of NF-κB. Compared to the positive control antibody 900389, the present antibody has a lower IC50 value and exhibits superior inhibitory effect.

[0126] Example 6 Nonspecific binding test (SPR) of humanized antibodies This test measures the nonspecific adsorption effect between antibody samples (900389 and HB0034) and non-target molecules using the SPR method. The reagents used in the test are shown in Table 13. [Table 13] The Series S Sensor Chip CM5 (GE, #BR-1005-30) chip was equilibrated at room temperature for 20-30 minutes and then set in a Biacore 8K (GE) instrument. Egg-derived lysozyme solution (Sigma, #L3790) and soy-derived trypsin inhibitor type 1-S (Sigma, #T-2327) were immobilized on channels 1 and 2 of the CM5 chip, respectively, using an amino group coupling kit (GE, #BR-1000-50). HBS-EP+(1X) (GE, #BR-1006-69) was used as the electrophoresis buffer, and five equilibration sacs were set up. Polyclonal rabbit anti-lysozyme (ABcam, #Ab391), anti-trypsin inhibitor antibody (LifeSpan Biosciences, #LS-C76609), chimeric antibody, and humanized antibody were each diluted to 1000 nM in electrophoresis buffer. A flow rate of 5 μL / min was set to injection channels 1, 2, and 3, and flow cells 1 and 2. The binding time was 10 min and the dissociation time was 15 min. The regeneration flow rate was set to 50 μL / min, and regeneration was performed for 60 seconds with 0.85% phosphoric acid solution (BIO-RAD, #176-2260), followed by regeneration for 30 seconds with 50 mM sodium hydroxide solution (GE, #BR-1003-58).

[0127] As a result, as shown in Table 14, the binding response value of BI control antibody 900389 to lysozyme exceeded 20 RU, but the binding response value to trypsin was less than 20 RU, indicating that the sample was negatively charged. In this test, the binding response value of 900389 to carboxymethyl dextran was 53.9 RU, indicating that the sample can bind to carboxymethyl dextran. Since the binding response values ​​of HB0034 to lysozyme and trypsin were all less than 20 RU, it is considered that the sample does not exhibit any obvious nonspecific electrostatic coupling. [Table 14] Note: After subtracting the effect of the buffer, it is generally considered that if the response value is less than 20 RU, the interaction is weak and negligible; if it is greater than 20 RU, there is a clear interaction; and if it is greater than 100 RU, there is a strong interaction.

[0128] Example 7: Therapeutic effect of humanized antibodies against psoriasis Cynomolgus monkeys were divided into five groups of six, each designated as a model control group, a positive control drug group, a low-dose HB0034 group, a medium-dose HB0034 group, and a high-dose HB0034 group. Psoriasis models were created by applying imiquimod cream (IMQ). On the first modeling day (Day 0) and on day 4 (Day 4), the low, medium, and high-dose HB0034 groups received intravenous injections of 5, 15, and 50 mg / kg of HB0034, respectively. The model control group received saline solution, and the positive control drug group received dexamethasone (5 mg / cm2, administered topically, once daily).

[0129] According to the study results, body weight decreased slightly in each group at the end of the experiment, and the HB0034 treatment group showed a tendency for body weight to decrease with increasing dose. Compared to saline (model group), at a dose of 5 mg / kg, HB0034 significantly reduced the levels of IL-17 and IL-36 in the modeled skin tissue (p<0.05) and showed a tendency for improvement in skin pathology scores, although the difference was not statistically significant (p>0.05) (see Table 15). At a dose of 15 mg / kg, HB0034 significantly improved the PASI score of the model (Study endpoint Day 10, p<0.05) and showed a tendency for improvement in skin pathology scores, although the difference was not statistically significant (p>0.05) (see Table 15, Figure 12). Levels of IL-17 and IL-36 in the modeled skin tissue were significantly reduced (p<0.05). At a dose of 50 mg / kg, HB0034 significantly improved the PASI score in the model (Days 4-10, p<0.05) and significantly improved skin pathology scores and skin IL-17 and IL-36 levels (see Table 15, Figures 11-12). Here, Figure 11 shows n=6, *p<0.05, **p<0.01 vs. saline, and Figure 12 shows n=6, **p<0.01 vs. saline.

[0130] Dexamethasone (5 mg / cm³) is a positive control drug. 2 The therapeutic effect of this model was slightly higher than that of HB0034 at 15 mg / kg, and slightly lower than that of HB0034 at 50 mg / kg.

[0131] As described above, at doses of 15-50 mg / kg (iv, Q4D × 2), HB0034 dose-dependently improves psoriasis-like symptoms of IMQ-induced erythema, dandruff, and skin thickening, reduces PASI scores, improves dermatopathological scores in modeling sites, and lowers levels of IL-17 and IL-36 in skin tissue. In the cynomolgus monkey IMQ-induced psoriasis model, the minimum effective dose of HB0034 is 15 mg / kg. [Table 15]

[0132] All documents relating to this application are cited as reference herein, so that each document may be cited independently. Furthermore, after reading the foregoing, a person skilled in the art may make various variations and modifications to the present invention, and it should be understood that equivalent forms of such variations are included within the scope of the claims of this application. This disclosure includes, for example, the embodiments described in the following sections. Section 1. (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and The heavy chain variable region containing the three complementarity-determining regions of CDR3 shown in SEQ ID NO:5, and / or (2) CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and An antibody characterized by containing a light chain variable region that includes three complementarity-determining CDRs of CDR3' as shown in SEQ ID NO:8. Section 2. The antibody according to item 1, characterized in that it is selected from animal antibodies, chimeric antibodies, humanized antibodies, or combinations thereof. Section 3. The heavy chain variable region sequence of the antibody is as shown in SEQ ID NO:1 or 9, and / or The antibody according to claim 1, characterized in that the light chain variable region sequence of the antibody is as shown in SEQ ID NO: 2, 10, 11, or 12. Section 4. (i) The antibodies described in item 1, and (ii) Recombinant protein comprising an optionally selected tag sequence to support expression and / or purification. Section 5. The scFV segment of the monoclonal antibody antigen-binding region is a binding region that specifically binds to IL-36R. The heavy chain variable region of the scFv includes, CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and The CDR3 shown in SEQ ID NO:5 includes three complementarity determination regions CDR, and / or The light chain variable region of the scFv includes, CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and A CAR construct characterized by containing three complementarity-determining regions of CDR3' shown in SEQ ID NO:8. Section 6. Recombinant immune cells characterized by expressing the exogenous CAR construct described in item 5. Section 7. (a) The antibody portion which is the antibody described in item 1, and (b) An antibody-drug conjugate characterized by comprising a binding site selected from the group consisting of a measurable marker, drug, toxin, cytokine, radionuclide, enzyme, or combination thereof. Section 8. The active portion is selected from the group consisting of the antibody described in item 1, the recombinant protein described in item 4, the CAR construct described in item 5, the immune cell described in item 6, the antibody-drug conjugate described in item 7, or a combination thereof, and the active component is used in (a) a measurement reagent or kit, and / or (b) the preparation of a pharmaceutical product for the prevention and / or treatment of IL-36-related disease. Section 9. The use according to item 8, characterized in that the aforementioned IL-36-related diseases include psoriasis. Section 10. (i) an active ingredient selected from the antibodies described in item 1, the recombinant proteins described in item 4, the CAR constructs described in item 5, the immune cells described in item 6, the antibody-drug conjugates described in item 7, or combinations thereof, and (ii) A pharmaceutical composition containing a pharmaceutically acceptable vector. Section 11. A polynucleotide characterized in that the polynucleotide code is the antibody described in item 1, the recombinant protein described in item 4, or the CAR construct described in item 5. Section 12. A vector characterized by containing the polynucleotide described in item 11. Section 13. A genetically modified host cell characterized by having the vector described in item 12 incorporated into the host cell, or the polynucleotide described in item 11 incorporated into the genome. Section 14. (1) In vitro, the sample comes into contact with the antibody described in item 1 or the antibody-drug conjugate described in item 7, (2) A method for non-diagnostic in vitro testing of IL-36R protein in a sample, comprising a procedure to measure whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of IL-36R protein in the sample. Section 15. A measurement plate characterized by comprising a plate (support plate) and a test piece containing the antibody described in item 1 or the antibody-drug conjugate described in item 7. Section 16. (1) A first container containing the antibody described in item 1, and / or (2) A second container containing a secondary antibody against the antibody described in item 1, Alternatively, a kit characterized by including the measuring plate described in item 15. Section 17. (a) Culture the host cells described in item 13 under conditions suitable for expression, (b) A method for preparing a recombinant polypeptide, comprising isolating a recombinant polypeptide from a culture, which is an antibody as described in item 1 or a recombinant protein as described in item 4.

Claims

1. (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and The heavy chain variable region containing the three complementarity-determining regions of CDR3 shown in SEQ ID NO:5, and (2) CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and An antibody targeting interleukin 36R, characterized by containing a light chain variable region that includes three complementarity-determining CDR regions of CDR3' as shown in SEQ ID NO:

8.

2. The antibody according to claim 1, characterized in that it is selected from animal antibodies, chimeric antibodies, humanized antibodies, and combinations thereof.

3. The heavy chain variable region and the light chain variable region are (1) Heavy chain variable region: SEQ ID NO:1, Light chain variable region: SEQ ID NO:2; (2) Heavy chain variable region: SEQ ID NO:9, Light chain variable region: SEQ ID NO:10; (3) Heavy chain variable region: SEQ ID NO:9, Light chain variable region: SEQ ID NO:11; and (4) Heavy chain variable region: SEQ ID NO:9, Light chain variable region: SEQ ID NO:12 The antibody according to claim 1, characterized by comprising a sequence selected from the group consisting of the following.

4. (i) the antibody according to claim 1, and (ii) A recombinant protein targeting interleukin 36R, comprising an optionally selected tag to support expression and / or purification.

5. The scFv segment of the antigen-binding region of a monoclonal antibody is a binding region that specifically binds to IL-36R. The heavy chain variable region of the scFv includes, CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and The CDR3 shown in SEQ ID NO:5 includes three complementarity determination regions CDR, and the light chain variable region of the scFv includes, CDR1' shown in SEQ ID NO:6, CDR2' shown in SEQ ID NO:7, and A CAR construct characterized by containing three complementarity-determining regions of CDR3' shown in SEQ ID NO:

8.

6. Recombinant immune cells characterized by expressing the CAR construct described in claim 5.

7. (a) an antibody portion which is the antibody described in claim 1, and (b) An antibody-drug conjugate comprising a binding site conjugated to the antibody portion, selected from the group consisting of measurable markers, drugs, toxins, cytokines, radionuclides, enzymes, and combinations thereof.

8. A pharmaceutical composition for use in the prevention and / or treatment of IL-36-related disease, comprising an active ingredient selected from the group consisting of the antibody described in claim 1, the recombinant protein described in claim 4, the CAR construct described in claim 5, the immune cell described in claim 6, the antibody-drug conjugate described in claim 7, and combinations thereof.

9. The pharmaceutical composition according to claim 8, characterized in that the IL-36-related disease includes psoriasis.

10. (i) an active ingredient selected from the antibody according to claim 1, the recombinant protein according to claim 4, the CAR construct according to claim 5, the immune cell according to claim 6, the antibody-drug conjugate according to claim 7, and combinations thereof, and (ii) A pharmaceutical composition containing a pharmaceutically acceptable carrier.

11. A polynucleotide encoding the antibody according to claim 1, the recombinant protein according to claim 4, or the CAR construct according to claim 5.

12. A vector characterized by comprising the polynucleotide described in claim 11.

13. A genetically engineered host cell comprising the vector according to claim 12, or the polynucleotide according to claim 11 incorporated into the genome.

14. (1) In vitro, the sample is brought into contact with the antibody described in claim 1 or the antibody-drug conjugate described in claim 7, (2) A method for non-diagnostic in vitro testing of IL-36R protein in a sample, comprising detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of IL-36R protein in the sample.

15. A measurement plate comprising a plate (support plate) and a test piece containing the antibody described in claim 1 or the antibody-drug conjugate described in claim 7.

16. A first container containing the antibody according to claim 1; A first container containing the antibody described in claim 1, and a second container containing a secondary antibody against the antibody described in claim 1; or, Measuring plate according to claim 15 A kit characterized by containing the following.

17. (a) Culturing the host cells described in claim 13 under conditions suitable for expression, (b) A method for preparing a recombinant polypeptide, comprising isolating a recombinant polypeptide from a culture, the recombinant polypeptide being the antibody described in claim 1 or the recombinant protein described in claim 4.

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