Antigen binding proteins targeting il-11

Abstract

Provided is an isolated antigen binding protein targeting IL-11. Also provided are methods of making and uses of the isolated antigen binding protein.

Description

Antigen binding protein targeting IL-11 Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an isolated antigen-binding protein targeting IL-11 and its use. Background Art

[0002] Interleukin-11 is a stromal cell-derived cytokine belonging to the IL-6 superfamily. It is secreted by various cell types, including osteoblasts, synovial cells, fibroblasts, chondrocytes, and trophoblasts. It then binds to IL-11R alpha and GP130 receptors and activates the downstream JAK / STAT signaling pathway.

[0003] More and more studies have found that IL-11 plays an extremely important role in cell protection, anti-inflammation and anti-fibrosis in organs such as the lungs. IL-11 is secreted by polarized cells or fibroblasts after injury and has autocrine and paracrine effects. In polarized cells, IL-11 causes cell dysfunction, initiates apoptotic cell death, and blocks regeneration. In stromal cells, IL-11 triggers extracellular matrix production, invasion and migration of myofibroblasts. IL-11-activated fibroblasts and myofibroblasts secrete cytokines and chemokines with strong pro-inflammatory effects. Inhibiting IL-11 can protect the parenchyma, resist fibrosis, and reduce matrix-driven inflammation. In tissues with regenerative capacity, inhibiting IL-11 can promote the proliferation and regeneration of damaged cells (such as hepatocytes) and organ regeneration.

[0004] In early 2020, Boehringer Ingelheim announced the acquisition of global exclusive rights to Enleofen's preclinical IL-11 platform to develop monoclonal antibody drugs targeting the IL-11 / IL-11Ra pathway to develop first-in-class therapies for the treatment of various fibroinflammatory diseases. LASN01, a monoclonal antibody drug targeting IL-11Ra developed by Lassen Therapeutics, is expected to be a new treatment for fibrosis and tumor cancer. A new drug research application will be submitted to the FDA in 2021 to initiate Phase I clinical trials. In China, the IL-11 monoclonal antibody (9MW3811) developed by Maiwei Biotechnology is at the forefront. It submitted a clinical trial application to the FDA in February 2023 and is currently in Phase I clinical trials.

[0005] As mentioned above, there are currently few studies targeting IL-11, and all of them are in the early research stage. No related drugs have been approved for marketing. Therefore, the development of drugs targeting this target has broad clinical application and treatment prospects. Summary of the Invention

[0006] The present application provides an isolated antigen-binding protein that targets IL-11 and has the following advantages: (1) having extremely strong affinity for human and / or animal IL-11, (2) being able to block IL-11-mediated activation of hIL-11 Effector Reporter Cell, and (3) being able to inhibit fibrosis.

[0007] In one aspect, the present application provides an isolated antigen-binding protein comprising at least one CDR in the heavy chain variable region VH or VHH.

[0008] In certain embodiments, the antigen binding protein comprises HCDR3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3.

[0009] In certain embodiments, the antigen binding protein comprises HCDR2, and the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2.

[0010] In certain embodiments, the antigen binding protein comprises HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1.

[0011] In certain embodiments, the antigen binding protein comprises HCDR3, HCDR2 and HCDR1, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1.

[0012] In certain embodiments, the antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 is as shown in SEQ ID NO:4.

[0013] In certain embodiments, the antigen binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is shown in SEQ ID NO:5.

[0014] In certain embodiments, the antigen binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is as shown in SEQ ID NO:6.

[0015] In certain embodiments, the antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is as shown in SEQ ID NO:7.

[0016] In certain embodiments, the antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, the amino acid sequence of H-FR1 is shown in SEQ ID NO:4, the amino acid sequence of H-FR2 is shown in SEQ ID NO:5, the amino acid sequence of H-FR3 is shown in SEQ ID NO:6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:7.

[0017] In certain embodiments, the antigen binding protein comprises an antibody heavy chain variable region VH or VHH, and the amino acid sequence of the VH or VHH is shown in SEQ ID NO:10.

[0018] In certain embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0019] In certain embodiments, the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0020] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a nanobody and / or a heavy chain antibody.

[0021] In certain embodiments, the antibody is a nanobody and / or a heavy chain antibody.

[0022] In certain embodiments, the antigen-binding fragment is a VHH.

[0023] In certain embodiments, the VHH comprises the amino acid sequence shown in SEQ ID NO:10.

[0024] In certain embodiments, the antigen binding protein comprises an antibody heavy chain constant region.

[0025] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9.

[0026] On the other hand, the present application also provides a chimeric antigen receptor, which comprises a targeting portion, and the targeting portion comprises the antigen binding protein.

[0027] On the other hand, the present application also provides a polypeptide comprising the antigen-binding protein.

[0028] On the other hand, the present application also provides an immunoconjugate comprising the antigen-binding protein and / or the polypeptide.

[0029] In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein and / or the chimeric antigen receptor.

[0030] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0031] On the other hand, the present application also provides a cell comprising the antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the nucleic acid molecule and / or the vector.

[0032] On the other hand, the present application also provides a method for preparing the antigen binding protein, the chimeric antigen receptor, and the polypeptide, which comprises culturing the cell under conditions such that the antigen binding protein, the polypeptide, and the chimeric antigen receptor are expressed.

[0033] On the other hand, the present application also provides a method for preparing modified immune cells, which comprises introducing the vector into immune cells.

[0034] On the other hand, the present application also provides a pharmaceutical composition comprising the antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and / or a pharmaceutically acceptable carrier.

[0035] On the other hand, the present application also provides a method for detecting the presence and / or content of IL-11 protein in a sample, which comprises using the antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0036] On the other hand, the present application also provides a detection kit for IL-11 protein, which comprises the antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0037] On the other hand, the present application also provides the use of the antigen binding protein, the chimeric antigen receptor, the polypeptide, and / or the cell in preparing a kit.

[0038] On the other hand, the present application also provides the antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the cell, and / or the pharmaceutical composition, which are used to prevent, alleviate and / or treat diseases and / or conditions. On the other hand, the present application also provides the use of the antigen binding protein, the chimeric antigen receptor, the polypeptide, the immunoconjugate, the cell, and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease or condition.

[0039] In certain embodiments, the medicament is for preventing, alleviating and / or treating a disease and / or condition.

[0040] In certain embodiments, the disease and / or disorder comprises an IL-11-associated disease.

[0041] In certain embodiments, the disease and / or condition comprises an inflammatory disease or a tumor.

[0042] In certain embodiments, the disease and / or condition comprises an IL-11-associated inflammatory disease or tumor.

[0043] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0045] FIG1 shows the effect curve of the anti-IL-11 antibody described in the present application in inhibiting the activation of hIL-11 Effector Reporter Cell mediated by IL-11.

[0046] FIG2 shows the PCR results of the anti-IL-11 antibody described in the present application for detecting the ability of the anti-IL-11 antibody to inhibit TGFβ-induced fibrosis in NICH0018 (skin fibroblasts of healthy individuals).

[0047] FIG3 shows the ELISA results of the anti-IL-11 antibody described in the present application for detecting the ability of the anti-IL-11 antibody to inhibit TGFβ-induced fibrosis in NICH0018 (skin fibroblasts of healthy humans). DETAILED DESCRIPTION

[0048] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0049] Definition of terms

[0050] In this application, the term "IL-11" generally refers to a stromal cell-derived cytokine that belongs to the IL-6 superfamily. The term "IL-11" encompasses "full-length," unprocessed IL-11 as well as any form of IL-11 produced by cell processing. In this application, the term "IL-11" includes mutants, fragments, variants, isoforms, and homologs thereof.

[0051] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0052] In the present application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained by artificial means from a natural state. The "isolated antigen-binding protein" may comprise a portion that binds to an antigen and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding portion to the antigen. The antigen-binding protein may comprise, for example, a protein framework region (FR) derived from an antibody or an alternative protein framework region or an artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions comprising, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions comprising, for example, biocompatible polymers. Examples of antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.

[0053] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structure-defining loop. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia, etc. These coding systems are known in the art. For example, the amino acid sequence numbering of the antigen-binding protein can be divided according to the IMGT numbering system. For example, the CDRs of the antigen-binding protein can be divided according to the Kabat numbering system.

[0054] In this application, the term "FR" generally refers to the more highly conserved portion of an antibody variable domain, which is referred to as a framework region. Typically, the variable domains of natural heavy and light chains each comprise four FR regions, i.e., four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0055] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L ” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0056] In this application, the term "VHH" is used interchangeably with "heavy chain antibody" and generally refers to an antibody that contains the variable antigen-binding domain of a heavy chain antibody. VHHs may also be referred to as nanobodies (Nb) and / or single-domain antibodies. For example, the VHH may bind to IL-11. For example, the VHH may be specific for IL-11.

[0057] In the present application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds to an antigen or target. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may include a signal peptide. The extracellular domain of CAR binds to the target antigen on the surface of the target cell, resulting in CAR clustering and transmitting the activation stimulus to the CAR-containing cell. CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of target antigen-expressing cells in a manner independent of major histocompatibility (MHC). For example, the extracellular structure may include the above-mentioned antigen binding protein. For example, the extracellular structure may specifically bind to IL-11.

[0058] Throughout this application, the terms "polypeptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked moieties. Each moiety can be a polypeptide with a distinct property. This property can be a biological property, such as in vitro or in vivo activity. It can also be a simple chemical or physical property, such as binding to a target molecule or catalysis of a reaction. The two moieties can be directly linked by a single peptide bond or through a peptide linker.

[0059] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0060] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect a host cell, allowing the genetic material elements it carries to be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0061] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original mother cell (in the form of total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described herein. Cells may be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells. In certain embodiments, cells are mammalian cells. In certain embodiments, mammalian cells are HEK293 cells.

[0062] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linked via a linker molecule) other agents (e.g., chemotherapeutic agents, radioactive elements, cytostatic agents, and cytotoxic agents) to the antibody or its antigen-binding fragment, which can deliver the other agents to target cells (e.g., tumor cells) through the specific binding of the antibody or its antigen-binding fragment to antigens on the target cells.

[0063] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0064] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0065] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0066] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0067] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to an IL-11 protein). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the change (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide prior to the change. For example, the substitution may be a conservative substitution.

[0068] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to an IL-11 protein or a fragment thereof).

[0069] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology percentage ratio " in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce the sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio can be realized by various ways known in the art.

[0070] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0071] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0072] Detailed Description of the Invention

[0073] Isolated antigen binding protein

[0074] In the present application, an isolated antigen binding protein is provided, which has one or more of the following characteristics: (1) having extremely strong affinity for human and / or animal IL-11, (2) being able to block IL-11-mediated activation of hIL-11 Effector Reporter Cell, and (3) being able to inhibit fibrosis.

[0075] In the present application, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof. In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb. In the present application, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

[0076] CDR

[0077] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR. In the present application, the isolated antigen-binding proteins are defined by the Kabat numbering system.

[0078] In the present application, the isolated antigen-binding protein may comprise at least one CDR in the antibody heavy chain variable region VH or VHH, the amino acid sequence of which is shown in SEQ ID NO: 10.

[0079] In the present application, the isolated antigen-binding protein may comprise HCDR3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3.

[0080] In the present application, the isolated antigen-binding protein may comprise HCDR2, and the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2.

[0081] In the present application, the isolated antigen-binding protein may comprise HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1.

[0082] In the present application, the isolated antigen-binding protein may comprise HCDR3, HCDR2 and HCDR1, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR is shown in SEQ ID NO: 1.

[0083] FR

[0084] In this application, the antibody framework region FR refers to the portion of the antibody variable region that is present between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide a framework for presenting six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface.

[0085] In the present application, the isolated antigen-binding protein may comprise H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 is as shown in SEQ ID NO:4.

[0086] In the present application, the isolated antigen-binding protein may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is shown in SEQ ID NO:5.

[0087] In the present application, the isolated antigen-binding protein may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is shown in SEQ ID NO: 6.

[0088] In the present application, the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO:7.

[0089] VH / VHH

[0090] In the present application, the isolated antigen-binding protein may comprise a variable region VH or VHH, and the amino acid sequence of the VH or VHH is shown in SEQ ID NO:10.

[0091] In the present application, the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0092] For example, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0093] For example, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0094] In the present application, the isolated antigen-binding protein may comprise VHH, and the amino acid sequence of VHH is shown in SEQ ID NO:10.

[0095] In the present application, the separated antigen-binding proteins may comprise an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the separated antigen-binding proteins may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In certain embodiments, the separated antigen-binding proteins may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region and subjected to amino acid mutations.

[0096] For example, the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO: 8 or SEQ ID NO: 9.

[0097] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy chain and / or light chain sequences that have one or more conservative sequence modifications thereto. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not eliminate antigen binding.

[0098] Chimeric antigen receptors, polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions

[0099] On the other hand, the present application also provides a chimeric antigen receptor (CAR), which may comprise a targeting portion that binds to IL-11.

[0100] In another aspect, the present application provides a polypeptide molecule, which may comprise the isolated antigen-binding protein described herein.

[0101] In certain embodiments, the polypeptide molecule may comprise a fusion protein. In certain embodiments, the polypeptide molecule may be a fusion protein.

[0102] In another aspect, the present application provides an isolated nucleic acid molecule that can encode an isolated antigen-binding protein described herein. For example, the nucleic acid molecule can be produced or synthesized by: (1) in vitro amplification, such as by polymerase chain reaction (PCR); (2) cloning and recombination; (3) purification, such as by enzymatic digestion and gel electrophoresis fractionation; or (4) synthesis, such as by chemical synthesis.

[0103] On the other hand, the present application provides a vector that can contain the nucleic acid molecules described herein. In addition, the vector can also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector can also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells so that the genetic material elements it carries are expressed in the host cells. The vector can include, for example, a plasmid, a cosmid, a virus, a bacteriophage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector can also include components that assist its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0104] On the other hand, the application provides a kind of cell, it can comprise the nucleic acid molecule described in the application or the vector described in the application.In certain embodiments, each kind or each host cell can comprise one or a kind of nucleic acid molecule or vector described in the application.In certain embodiments, each kind or each host cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecules or vector described in the application.For example, the vector described in the application can be introduced into the host cell, for example, eukaryotic cell, such as cell, fungus or yeast cell from plant etc.In certain embodiments, the cell can be bacterial cell (for example, Escherichia coli), yeast cell or other eukaryotic cell, for example COS cell, Chinese hamster ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2 / 0 cell, NS0 cell or myeloma cell.The vector described in the application can be introduced into the host cell by methods known in the art, for example, electroporation, lipofectine transfection, lipofectamin transfection etc.

[0105] On the other hand, the present application also provides an immunoconjugate, which may comprise the isolated antigen-binding protein described in the present application.

[0106] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0107] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0108] In certain embodiments, the pharmaceutical composition may be a bispecific or multispecific molecule comprising the isolated antigen-binding protein described herein. In certain embodiments, in addition to the molecule that binds IL-11, the other molecules in the bispecific or multispecific molecule may bind to targets unrelated to IL-11.

[0109] In certain embodiments, the pharmaceutical composition may also contain more than one active compound, generally those having complementary activities that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

[0110] In certain embodiments, the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe and non-toxic.

[0111] Preparation method

[0112] In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may include culturing the host cell described herein under conditions that allow for expression of the antigen-binding protein. For example, the method may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

[0113] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding proteins of the present application. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes. For example, a hybridoma preparation method can be used to obtain spleen cells from immunized mice and fuse them with SP2 / 0 myeloma cells, and then screen the hybridoma cell line using HAT.

[0114] Any suitable form of IL-11 can be used as an immunogen (antigen) to generate non-human antibodies specific for IL-11 and screen for biological activity of the antibodies. For example, the stimulating immunogen can be full-length mature human IL-11, including natural homodimers, or peptides containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0115] Chimeric human antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody can be an IgG antibody, and the IgG1 subtype can be used. The optimization of the necessary constant domain sequence can be achieved by screening the antibody using the biological assays described in the examples below to produce the desired biological activity. Similarly, any class of light chain can be used in the compounds and methods of the present application. For example, a kappa chain or a variant thereof can be used in the compounds and methods of the present application.

[0116] Methods and uses

[0117] On the other hand, the present application provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0118] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, which may comprise administering the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein to a subject in need thereof.

[0119] On the other hand, the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein can be used to prevent and / or treat diseases and / or disorders.

[0120] In the present application, the disease and / or disorder may be an IL-11-related disease and / or disorder.

[0121] In the present application, the disease and / or disorder may be an inflammatory disease or a tumor.

[0122] In the present application, the diseases and / or disorders include IL-11-related inflammatory diseases or tumors.

[0123] On the other hand, the present application also provides a method for detecting IL-11 in a sample, which comprises administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0124] In some cases, the method for detecting IL-11 in a sample is an in vitro method. In some cases, the method for detecting IL-11 in a sample is for non-therapeutic purposes. In some cases, the method for detecting IL-11 in a sample is not a diagnostic method.

[0125] On the other hand, the present application also provides a reagent or kit for detecting IL-11 in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0126] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in preparing a kit for detecting the presence and / or content of IL-11 in a sample.

[0127] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0128] Example

[0129] Example 1 Preparation of Alpaca Monoclonal Antibody Against Human IL-11 - Original Antibody

[0130] Unimmunized alpacas were selected, and 5 mL of blood was collected before immunization to separate the serum as a negative control. IL-11 Protein, Human, Recombinant (Sino Biological Inc., Cat No: 12225-HNCE) was used as the antigen. 0.5 mg of the antigen was fully emulsified with an equal volume of Freund's complete adjuvant (Sigma, Cat No: F5881) and injected subcutaneously at multiple points. Subsequently, every three weeks, a 0.25 mg dose of antigen was fully emulsified with incomplete Freund's adjuvant (Sigma, Cat No.: F5506) and the alpacas were immunized three times subcutaneously at multiple points. 50 mL of peripheral blood was collected one week after the third and fourth immunizations for antibody titer titration and nanoantibody library construction. The serum after the third and fourth immunizations was titered, and the serum antibody titer after the third immunization was >32,000, and the serum antibody titer after the fourth immunization was >64,000, both meeting the library construction requirements.

[0131] Example 2 Construction and screening of immune library

[0132] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood collected after three or four immunizations of alpacas. After pooling, total RNA was extracted and reverse-transcribed into cDNA. VHH3, VHH4, and VH-like VHH gene segments were amplified using a one-step PCR method. The VHH gene segments were cloned into pShort phagemids, and the resulting phagocyte-mediated gene transfer reaction products were electroporated into SR320 supernatant cells containing a helper phage. After expansion and culture, the phage was harvested to form the immune library. The quality data of the immune library are shown in Table 1. The culture supernatant was collected to obtain the antibody library (Phage), which was then aliquoted and stored frozen at -80°C.

[0133] Table 1 Statistics of related indicators of alpaca immune library Lib11-VHHall

[0134] Example 3 Biopanning of Antibody Library and Protein Expression

[0135] Through solid-phase protein screening, using human IL-11 recombinant protein as bait, multiple rounds of protein panning were performed to achieve enrichment. High-affinity positive clones were screened using phage ELISA. Single clone sequencing identified 11 unique clones, which were divided into five groups based on differences in CDRH3 amino acid sequences. The phage enriched with human IL-11 was subjected to next-generation sequencing (NGS). Of the 20 sequences identified by NGS, 17 were successfully expressed in eukaryotic cells.

[0136] Example 4 Preparation of 700068 Heavy Chain Antibody

[0137] 4. Preparation of 1400208 plasmid (SEQ ID NO: 11)

[0138] A signal peptide sequence was added to the obtained antibody variable region gene before gene synthesis. The target gene was ligated into a mammalian cell expression vector containing human Fc through HindIII and BamHI restriction sites. Leucine L234 and Leucine L235 (EU numbering) in the CH2 region of the Fc on this vector were mutated to alanine A. These Fc mutations abolished Fc binding to FcγRs and C1q without affecting FcRn binding, thereby significantly reducing the antibody's ADCC / CDC effects. After ligation, the complete expression vector was obtained, designated as 400208.

[0139] The 400208 expression vector was amplified in Escherichia coli, and sufficient plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd., Cat. No. DP117) and sequenced to confirm that the plasmid was completely consistent with the theoretical sequence.

[0140] 4.2 Antibody Expression

[0141] The prepared plasmid was transiently transfected into host CHO-S cells and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity column to obtain antibody 700068.

[0142] 700068-VHH (SEQ ID NO: 10)

[0143] Note: The italic, underlined and bold parts are the CDR regions under Kabat numbering.

[0144] 700068-Fc (SEQ ID NO: 8)

[0145] Example 5 700077 Antibody Preparation

[0146] 5.1 Preparation of 400231 plasmid (SEQ ID NO: 12)

[0147] Using the expression plasmid for antibody 700068 as a template, PCR amplification was performed to obtain the target gene fragment. PCR primers and sequences are shown in Table 1. The mammalian cell expression vector containing the human light chain constant region was digested with the restriction endonucleases HindIII / EcoRI and ligated to the target gene fragment. This complete expression vector was obtained, designated 400231.

[0148] Table 2 PCR primers and sequences

[0149] The 400231 expression vector was amplified by E. coli and Sufficient plasmid was prepared using the Xtra Maxi Plus EF Plasmid Extraction Kit (MACHEREY-NAGEL, Cat. No. 740426.50). The extracted plasmid was digested with the restriction endonuclease pvuI (NEB, Cat. No. R0150L). After digestion, the linearized plasmid was precipitated with ethanol and sequenced to confirm that the plasmid was identical to the theoretical sequence.

[0150] 5.2 Expression of 700077 Antibody

[0151] The linearized plasmid 400231 was stably transfected into the host cell CHO-K1 for expression. After culture for a period of time, the cell culture supernatant was collected by centrifugation and purified using a Protein A affinity column to obtain antibody 700077.

[0152] 700077-VHH (SEQ ID NO: 10)

[0153] Note: The italic, underlined and bold parts are the CDR regions under Kabat numbering.

[0154] 700077-Fc (SEQ ID NO: 9)

[0155] Example 6 Affinity Detection of 700068 Heavy Chain Antibody to Human and Mouse IL-11

[0156] The SPR method was used to determine the binding kinetic constants of human and mouse IL-11 and antibodies. The experimental materials and instruments are as follows:

[0157] The sample chamber and flow cell temperature of the Biacore T200 were set to 25°C, and the data collection frequency was 10 Hz. Antibody samples were captured to 300 RU on a Series S Protein A chip as ligands. Human and mouse IL-11 antigens were diluted with HBS-EP+, pH 7.4 buffer to a series of gradient concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.56 nM, 0.78 nM, and 0.39 nM as analytes, and HBS-EP+, pH 7.4 buffer was used as the zero concentration for background subtraction. A multi-cycle kinetic method was used with an analyte flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 600 s, and a regeneration of 10 mM glycine (pH 1.5) at 50 μL / min for 60 s. Data were analyzed in 1:1 binding mode and Fit Local Kinetics mode.

[0158] The experimental results are shown in Table 3:

[0159] The affinity constants (KD / M) for various species showed that the affinity of the anti-IL-11 monoclonal heavy chain antibody 700068 of the present invention for human and mouse IL-11 was 10 -09 It is on the order of M and has extremely strong affinity for human and mouse antigens.

[0160] Table 3 Affinity test results of 700068 heavy chain antibody to human and mouse IL-11

[0161] Note: NB stands for Not Binding

[0162] Example 7 Affinity Detection of 700068 Heavy Chain Antibody and Rat IL-11

[0163] The BLI method was used to determine the binding kinetic constants of rat IL-11 and antibody. The experimental materials and instruments are as follows:

[0164] Kinetic analysis was performed using an SA sensor loaded with 5 μg / mL biotinylated sample as the ligand and a gradient-diluted Rat IL-11 solution as the analyzer. Kinetic constants were analyzed using a 30°C temperature, 1000 rpm shaking rate, 360 s association, 360 s dissociation, and a 1:1 binding model global fit.

[0165] The experimental results are shown in Table 4:

[0166] The affinity constants (KD / M) of the humanized anti-IL-11 monoclonal antibody 700068 of the present invention for rat IL-11 were 10 -09 M order of magnitude, with extremely strong affinity for rat antigens.

[0167] Table 4 Affinity test results of 700068 heavy chain antibody and rat IL-11

[0168] Example 8 Affinity Detection of 700068 Monoclonal Heavy Chain Antibody and Monkey IL-11

[0169] The SPR method was used to determine the monkey antigen-antibody binding kinetic constants. The experimental materials and instruments are as follows:

[0170] The sample chamber and flow cell temperature of the Biacore 8k were set at 25°C, and the data collection frequency was 10 Hz. Cynomolgus IL-11 was immobilized by amino coupling on a Series S Sensor Chip CM5 using the Amine Coupling Kit. Monkey IL-11 was diluted in HBS-EP+, pH 7.4 buffer to a gradient concentration of 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM as the analyte, with HBS-EP+, pH 7.4 buffer used as the zero concentration for background subtraction. A single-cycle kinetic method was used with an analyte flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 600 s, and a regeneration time of 10 mM glycine (pH 1.5) at 50 μL / min for 60 s. Data were analyzed in 1:1 binding mode and Fit Local Kinetics mode.

[0171] The experimental results are shown in Table 5:

[0172] The affinity constant (KD / M) results showed that the affinity of the anti-IL-11 heavy chain antibody 700068 of the present invention to monkey IL-11 was 10 -09 M order of magnitude, with extremely strong affinity for monkey antigens.

[0173] Table 5 Affinity test results of 700068 heavy chain antibody and monkey IL-11

[0174] Example 9 Non-specific binding test of 700068 monoclonal heavy chain antibody

[0175] The Biacore 8k sample chamber and flow path temperature were set at 25°C. Lysozyme solution from chicken egg and trypsin inhibitor type 1-S from Glycine max were amino-coupled to a CM5 chip as ligands. The antibody was diluted to 1 μM, flow rate: 10 μL / min, association: 900 s, dissociation: 1200 s. Regeneration was performed first with 0.85% phosphoric acid at a flow rate of 50 μL / min for 30 s, followed by regeneration with 50 mM sodium hydroxide at a flow rate of 50 μL / min for 30 s. Binding stability signals were collected to analyze nonspecific electrostatic binding of the antibody.

[0176] Quality control results for lysozyme solution from chicken egg and trypsin inhibitor type 1-S from Glycine max using a polyclonal rabbit anti-lysozyme and anti-trypsin inhibitor antibody, respectively, confirmed that the activity of egg white lysozyme and soybean trypsin inhibitor after amino-coupling immobilization on a CM5 chip was normal, indicating that the immobilized ligand activity was maintained throughout the experiment. The PI of trypsin inhibitor type 1-S from Glycine max was 4.5, and that of lysozyme was 11.3. In the HBS-EP buffer system at pH 7.4, trypsin inhibitor type 1-S from Glycine max has a strong negative charge, while lysozyme has a strong positive charge.

[0177] The experimental results are shown in Table 6:

[0178] The binding response values ​​of all tested samples with Lysozyme and Trypsin were less than 20RU, indicating that the tested samples had no obvious nonspecific electrostatic binding effect.

[0179] Table 6 Nonspecific binding data of 700068 heavy chain antibody

[0180] Example 10 Anti-IL-11 Monoclonal Heavy Chain Antibody (700068) Blocks IL-11-Mediated Activation of hIL-11 Effector Reporter Cells

[0181] hIL-11 Effector Reporter Cells (Nanjing Kebai Biotechnology, Cat#: CBP74114) were collected and resuspended in DMEM medium (Gibco, Cat#: 11965092) containing 10% FBS (Gibco, Cat#: 10099141C) to dilute the cells to 1.2×10 6 cells / mL, 50 μL was added to each well of a 96-well plate (Corning, Cat#: 3599) and incubated overnight in a 37°C, 5% CO2 incubator. Anti-IL-11 antibody was diluted to 120 μg / mL in DMEM medium supplemented with 10% FBS and then serially diluted three-fold to 10 concentrations, for a total of 11 concentrations. 25 μL of each antibody concentration was added to each well of the 96-well plate. Human IL-11 (novoprotein, Cat#: C006) was diluted to 1 ng / mL in DMEM medium supplemented with 10% FBS and added to each well of the 96-well plate at 25 μL. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, the 96-well plate was removed and allowed to equilibrate at room temperature for 15 minutes. Bright-Glo reagent (Promega, Cat#: E2620) was added to each well at 100 μL and the plate was read using a microplate reader (MD i3x) for full-wavelength fluorescence detection.

[0182] The experimental results are shown in Figure 1:

[0183] The anti-IL-11 heavy chain antibody 700068 of the present invention can block the activation of hIL-11 Effector Reporter Cell mediated by IL-11.

[0184] Example 11 Ability of IL-11 Heavy Chain Antibody to Inhibit Fibrosis in Vitro

[0185] The ability of anti-IL-11 heavy chain antibody 700077 to inhibit TGFβ (transforming growth factor β)-induced fibrosis in NICH0018 (skin fibroblasts from healthy individuals) was tested. After removing the culture medium (89% DMEM (Dulbecco's medium) + 10% FBS (fetal bovine serum) + 1% P / S (penicillin, streptomycin)) from the NICH0018 cells in the logarithmic growth phase in the culture flask, the cells were washed twice with 10 mL of PBS, and 2 mL of 0.25% trypsin was added. The cells were digested in an incubator (37°C, 5% carbon dioxide) for 3-5 minutes. After adding culture medium to terminate the digestion, the cell suspension was obtained, centrifuged (500g×5min), the culture medium was discarded, and the cells were resuspended to 1 mL with culture medium and counted on a counter. 5×10 cells per well were used. 5Cells were plated into six-well plates and supplemented with medium to 2 mL per well. After 8 hours of incubation, the cells were starved for 16 hours after attachment. The culture medium was then replaced with complete medium (DMEM without FBS). The culture medium was prepared to a final concentration of 10 ng / mL TGFβ or 15 μg / mL 700077. After aspirating the original medium, 2 mL of standard culture medium was added to the blank control group, 2 mL of culture medium containing 10 ng / mL TGFβ was added to the stimulation group, and 2 mL of culture medium containing a final concentration of 10 ng / mL TGFβ and 15 μg / mL 700077 was added to the antibody control group. Three replicates were used for each group. After 24 hours of incubation in the incubator, the supernatant was collected (frozen at -20°C for subsequent ELISA analysis), washed twice with PBS, and the cells were harvested using a cell scraper into an RNase-free EP tube. The cell pellet was then centrifuged (500 g for 5 minutes).

[0186] RT-PCR was used to detect the expression of the fibrosis-related genes FN (encoding fibronectin) and COL1A1 (encoding type I collagen alpha 1) in cells. Add 350 μL of Buffer RLT to the cell pellet and mix thoroughly with the cells to lyse them. Add 350 μL of 70% ethanol to the lysate and mix thoroughly. Transfer the previous lysate to a 2 mL collection tube containing an RNeasy MinElute spin column, centrifuge at 8000 g for 15 seconds, and discard the filtrate. Add 350 μL of Buffer RW1 to the RNeasy MinElute spin column, centrifuge at 8000 g for 15 seconds, and discard the filtrate. Place the RNeasy MinElute spin column in a new 2 mL collection tube, add 500 μL of Buffer RPE, centrifuge at 8000 g for 15 seconds, and discard the filtrate. Add 500 μL of 80% ethanol to the RNeasy MinElute spin column, centrifuge at 8000 g for 2 minutes, and discard the collection tube. Place the RNeasy MinElute spin column in a new 2 mL collection tube. With the cap open, centrifuge at maximum speed for 5 minutes. Discard the filtrate and collection tube. Place the RNeasy MinElute spin column in a new 1.5 mL collection tube. Add 14 μL of RNase-free water to the center of the column. Centrifuge at maximum speed for 1 minute. Elute the RNA and measure the RNA concentration.

[0187] The genome removal reaction system and reaction conditions are as follows:

[0188] After obtaining the reaction solution without the reaction group, reverse transcription reaction was performed. The reaction system and reaction conditions were as follows:

[0189] After obtaining cDNA, perform quantitative PCR:

[0190] Prepare the reaction mixture according to the table below and add 11.2 μL of the mixture to the corresponding reaction tubes. Mix 2 μL of sample cDNA with 6.8 μL of DEPC HO and add it to the corresponding wells. Also set up a negative control well with sterile water as the template.

[0191] Programming: Use the comparative CT (ΔΔCT) method and set up the software to run the PCR amplification program: denaturation at 95°C for 10 minutes, 1 cycle; denaturation at 95°C for 15 seconds, extension at 60°C for 30 seconds, 40 cycles; denaturation at 95°C for 15 seconds, extension at 60°C for 1 minute, with a 0.15°C gradient ramp to 95°C. Run quantitative PCR.

[0192] The experimental results are shown in Figure 2:

[0193] The anti-IL-11 heavy chain antibody 700077 of the present invention can inhibit the increase of FN and COL1A1 induced by TGFβ.

[0194] The experimental results are shown in Figure 3:

[0195] The results of FN gene expression were verified at the protein level by ELISA (enzyme-linked immunosorbent assay), demonstrating that 700077 could inhibit TGFβ-induced fibrosis in NICH0018 cells.

[0196] Note: In Figures 2 and 3, data are expressed as mean ± SEM, n = 3. *P ≤ 0.05, ***P ≤ 0.001, analyzed by one-way ANOVA.

Claims

1. An isolated antigen binding protein targeting IL-11, comprising HCDR3, HCDR2 and HCDR1 of a heavy chain variable region VH or VHH; wherein, The amino acid sequence of the HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:

1.

2. The antigen binding protein according to claim 1, comprising H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 is as shown in SEQ ID NO:

4.

3. The antigen binding protein according to any one of claims 1-2, comprising H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is as shown in SEQ ID NO:

5.

4. The antigen binding protein according to any one of claims 1 to 3, comprising H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is as shown in SEQ ID NO:

6.

5. The antigen binding protein according to any one of claims 1 to 4, comprising H-FR4, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is as shown in SEQ ID NO:

7.

6. The antigen-binding protein according to any one of claims 1 to 5, comprising an antibody heavy chain variable region VH, and the amino acid sequence of the VH is shown in SEQ ID NO:

10.

7. The antigen binding protein according to any one of claims 1 to 6, which comprises an antibody or an antigen binding fragment thereof.

8. The isolated antigen-binding protein of claim 7, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

9. The antigen binding protein according to any one of claims 7-8, wherein the antigen binding fragment is a VHH.

10. The antigen binding protein according to any one of claims 1 to 9, wherein the amino acid sequence of the VHH is shown in SEQ ID NO:

10.

11. The antigen binding protein according to any one of claims 7 to 10, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a single-chain antibody, a nanobody and / or a heavy chain antibody.

12. The antigen binding protein according to claim 11, wherein the antibody is a nanobody and / or a heavy chain antibody.

13. The antigen binding protein according to any one of claims 1 to 12, comprising an antibody heavy chain constant region.

14. The antigen binding protein according to any one of claims 1 to 13, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

15. The antigen binding protein according to any one of claims 1 to 14, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

16. The antigen binding protein according to any one of claims 1 to 15, wherein the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO: 8 or SEQ ID NO:

9.

17. A chimeric antigen receptor comprising a targeting moiety comprising the antigen binding protein of any one of claims 1-16.

18. A polypeptide comprising the isolated antigen binding protein of any one of claims 1 to 16 or the chimeric antigen receptor of claim 16.

19. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-16.

20. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1-16, the chimeric antigen receptor of claim 17, or the polypeptide molecule of claim 18.

21. A vector comprising the nucleic acid molecule according to claim 20.

22. A cell comprising the isolated antigen binding protein of any one of claims 1-16, the chimeric antigen receptor of claim 17, the polypeptide molecule of claim 18, the immunoconjugate of claim 19, the nucleic acid molecule of claim 20, or the vector of claim 21.

23. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1 to 16, the chimeric antigen receptor of claim 17, the polypeptide molecule of claim 18, the immunoconjugate of claim 19, the nucleic acid molecule of claim 20, the vector of claim 21 and / or the cell of claim 22, and optionally a pharmaceutically acceptable carrier.

24. A method of making the isolated antigen binding protein of any one of claims 1-16, the method comprising culturing the cell of claim 22 under conditions such that the antigen binding protein is expressed.

25. Use of the isolated antigen binding protein of any one of claims 1 to 16, the chimeric antigen receptor of claim 17, the polypeptide molecule of claim 18, the immunoconjugate of claim 19, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 and / or the pharmaceutical composition of claim 23 in the preparation of a medicament for preventing, alleviating and / or treating a disease and / or condition.

26. The use according to claim 25, wherein the disease and / or disorder comprises an IL-11 related disease.

27. Use according to claim 25 or 26, wherein the disease and / or disorder comprises an inflammatory disease or a tumor.

28. A method for detecting IL-11 in a sample, the method comprising administering the isolated antigen binding protein of any one of claims 1 to 16, the chimeric antigen receptor of claim 17, the polypeptide molecule of claim 18, the immunoconjugate of claim 19, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 and / or the pharmaceutical composition of claim 23.

29. A reagent or kit for detecting IL-11 in a sample, comprising the isolated antigen binding protein of any one of claims 1 to 16, the chimeric antigen receptor of claim 17, the polypeptide molecule of claim 18, the immunoconjugate of claim 19, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 and / or the pharmaceutical composition of claim 23.

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