WO2023016505A1 - Il-11人源化抗体及其应用 - Google Patents

Il-11人源化抗体及其应用 Download PDF

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Publication number
WO2023016505A1
WO2023016505A1 PCT/CN2022/111608 CN2022111608W WO2023016505A1 WO 2023016505 A1 WO2023016505 A1 WO 2023016505A1 CN 2022111608 W CN2022111608 W CN 2022111608W WO 2023016505 A1 WO2023016505 A1 WO 2023016505A1
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antibody
seq
antigen
amino acid
heavy chain
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French (fr)
Inventor
周加滔
陈苍沙
王银彩
介淼星
董军纪
李文佳
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Sunshine Lake Pharma Co Ltd
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Sunshine Lake Pharma Co Ltd
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Priority to JP2024506768A priority Critical patent/JP2024532709A/ja
Priority to US18/293,271 priority patent/US20250002574A1/en
Priority to EP22855485.3A priority patent/EP4386002A4/en
Publication of WO2023016505A1 publication Critical patent/WO2023016505A1/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/33—Antibodies; T-cell engagers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of biotechnology, specifically, the present invention relates to antibodies to IL-11 and applications thereof, more specifically, the present invention relates to antibodies capable of specifically recognizing IL-11 or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, Recombinant cells, pharmaceutical compositions, pharmaceutical uses, kits for detecting IL-11 and uses for preparing detection or diagnostic kits.
  • Fibrosis is an important process that is a critical part of wound healing. Excessive fibrosis is common in many rare and common diseases and is important in disease pathogenesis. Diseases characterized by excessive fibrosis include, but are not limited to: systemic sclerosis, scleroderma, hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), atrial fibrillation, ventricular fibrillation, myocarditis, cirrhosis, Kidney disease, eye disease, asthma, cystic fibrosis, arthritis, and idiopathic pulmonary fibrosis.
  • DCM dilated cardiomyopathy
  • interleukin 11 The true physiological role of interleukin 11 (IL-11) is unclear. IL-11 is most closely associated with hematopoietic cell activation and platelet production, but has also been found to be pro- and anti-inflammatory, pro-angiogenic, and important for neoplasia. IL-11 expression is known to be induced by TGF ⁇ 1 or tissue injury. From the published literature, the role of IL-11 in fibrosis is unclear. IL-11 is thought to be important for lung fibrosis and inflammation, and its expression levels correlate with collagen levels in the skin and respiratory system. However, most studies have shown that IL-11 is anti-fibrotic in the heart and kidney and anti-inflammatory in several tissues and chronic inflammatory diseases. In general, the molecular mode of action of IL-11 is considered to be through STAT3-mediated transcriptional regulation of mRNA levels of RNA expression.
  • Agents capable of inhibiting the action of IL-11 can prevent or reduce the binding of IL-11 to the IL-11 receptor.
  • IL-11 interleukin 11
  • the present invention develops a humanized Anti-IL-11 antibody with high neutralization activity, which is expected to be used for the treatment or prevention of fibrosis-related diseases.
  • the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically recognizing IL-11.
  • the antibody contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity therewith: heavy chain variable region CDR sequence: SEQ ID NO: 1-33, light chain Variable region CDR sequence: SEQ IN NO: 34-66, the antibody or its antigen-binding fragment has humanized modification.
  • the "humanized modification” described in this application refers to amino acid changes that can reduce the immunogenicity of the antibody or its antigen-binding fragment, including amino acid mutations, insertions, deletions, chemical groups superfluous and so on.
  • the immunogenicity of the above humanized antibody according to the embodiment of the present invention is reduced, and the antibody can specifically target and bind IL-11, and block the binding of IL-11 and IL-11 receptor.
  • the above antibody or antigen-binding fragment may further include at least one of the following additional technical features:
  • the antibody comprises: a heavy chain variable as shown in SEQ ID NO: 4, 5 and 6 or an amino acid sequence having at least 95% identity to SEQ ID NO: 4, 5 and 6, respectively Region CDR1, CDR2, CDR3 sequence.
  • the antibody comprises: a variable light chain as shown in SEQ ID NO: 37, 38 and 39 or an amino acid sequence having at least 95% identity with SEQ ID NO: 37, 38 and 39, respectively Region CDR1, CDR2, CDR3 sequence.
  • the antibody or antigen-binding fragment thereof specifically recognizes IL-11.
  • the antibody contains at least one of a heavy chain framework region sequence and a light chain framework region sequence, and at least a part of at least one of the heavy chain framework region sequence and the light chain framework region sequence is from a mouse At least one of antibody of human origin, antibody of primate origin, or mutant thereof.
  • the heavy chain framework region includes FL1, FL2, FL3 and FL4, and the heavy chain framework region FL1, FL2, FL3 and FL4 have SEQ ID NO: 67, 68, 69 and 70 respectively the amino acid sequence of
  • X1 is Q or H
  • X2 is M or I
  • X3 is V or A
  • X4 is M or L
  • X5 is R or A
  • X6 is Y or F.
  • the light chain framework region includes FL1, FL2, FL3 and FL4, and the light chain framework region FL1, FL2, FL3 and FL4 have SEQ ID NO: 71, 72, 73 and 74 respectively the amino acid sequence of
  • X7 is Y or F
  • X8 is V or M
  • X9 is Y or F.
  • the antibody has a heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO: 75-78.
  • the antibody has a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 79-82.
  • the antibody contains at least one of the heavy chain constant region and the light chain constant region, and at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from a murine antibody, human At least one of antibodies derived from primates, antibodies derived from primates, or mutants thereof.
  • the full-length sequence of the constant region of the antibody is shown in SEQ ID NO: 83 or 84.
  • the antibody has a heavy chain with an amino acid sequence shown in any one of SEQ ID NO:85-88 and a light chain with an amino acid sequence shown in any one of SEQ ID NO:89-92.
  • the antibody is a single-chain antibody, a multimeric antibody, a CDR-grafted antibody or a small molecule antibody.
  • the small molecule antibody includes at least one of Fab antibody, Fv antibody, single domain antibody and minimal recognition unit.
  • the invention proposes a nucleic acid molecule.
  • the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment encoded by the nucleic acid molecule according to the embodiment of the present invention has low immunogenicity, can specifically target and bind IL-11, and block the binding of IL-11 and IL-11 receptor.
  • the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:
  • the nucleic acid molecule is DNA.
  • the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO: 93-96 or has a nucleotide sequence shown in any one of SEQ ID NO: 97-100.
  • the present invention provides an expression vector.
  • the expression vector carries the aforementioned nucleic acid molecule.
  • the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the antibody or antigen-binding fragment thereof that specifically recognizes IL-11 as described above can be effectively realized, thereby realizing the described In vitro production of antibodies or antigen-binding fragments in large quantities.
  • the above-mentioned expression vector may further include at least one of the following additional technical features:
  • the expression vector is a eukaryotic expression vector. Furthermore, the expression of the aforementioned antibody specifically recognizing IL-11 or its antigen-binding fragment in eukaryotic cells, such as CHO cells, is realized.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the aforementioned nucleic acid molecule, or expresses the aforementioned antibody or antigen-binding fragment thereof.
  • the recombinant cells according to the embodiments of the present invention can be used for in vitro expression and mass production of the aforementioned antibody specifically recognizing IL-11 or an antigen-binding fragment thereof.
  • the above-mentioned recombinant cells may further include at least one of the following additional technical features:
  • the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.
  • the expression vector is introduced into the host cell by electrotransduction.
  • the recombinant cells are eukaryotic cells.
  • the recombinant cells are mammalian cells.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition contains the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell.
  • the antibody contained or expressed in the pharmaceutical composition according to the embodiment of the present invention can specifically target IL-11 and block the binding of IL-11 and IL-11 receptor.
  • the present invention proposes the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell, and the aforementioned pharmaceutical composition in the preparation of drugs
  • the use of said medicine is used to treat or pulmonary fibrosis (Pulmonary fibrosis), non-alcoholic steatohepatitis (non-alcoholic steatohepatitis, NASH), chronic heart failure (Chronic heart failure, CHF) or from fibroblast to fibrosis Diseases resulting from transformation.
  • the present invention provides a kit for detecting IL-11.
  • the kit includes the aforementioned antibody.
  • the aforementioned IL-11 antibody can specifically target and bind IL-11.
  • the kit according to the embodiment of the present invention can realize the specific detection of IL-11. For example, when the antibody is bound to a fluorescent group, fluorescence detection can be used The device realizes the localization or real-time detection of IL-11.
  • the present invention proposes the use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell in the preparation of a kit, the kit For detecting IL-11 or diagnosing IL-11 related diseases.
  • Figure 1 is the results of the preferred antibody competitively blocking the binding of IL-11 and IL-11R according to an embodiment of the present invention
  • Fig. 2 is the preferred antibody competitive blocking activity detection result according to the embodiment of the present invention.
  • Fig. 3 is the result of western detection that the preferred antibody strain according to the embodiment of the present invention inhibits lung fibroblast HFL-1 from transforming to fibrosis;
  • Fig. 4 is the results of statistical analysis of the inhibition of lung fibroblast HFL-1 to fibrosis transformation by preferred antibody strains according to an embodiment of the present invention
  • Figure 5 and Figure 6 are the results of the inhibition of pulmonary fibrosis in C57BL/6 mice by preferred antibodies according to the embodiments of the present invention.
  • Figure 7 is a graph showing the thermal stability test results of a preferred humanized antibody according to an embodiment of the present invention.
  • Figure 8 shows the competitive blocking activity of humanized antibodies according to an embodiment of the present invention.
  • Fig. 9 is the lung inflammatory injury score of SD rats after humanized antibody treatment according to an embodiment of the present invention.
  • Fig. 10 is the lung fibrosis score of SD rats treated with humanized antibodies according to an embodiment of the present invention.
  • Fig. 11 is the binding activity of an affinity matured antibody according to an embodiment of the present invention.
  • Figure 12 is the competitive blocking activity of affinity matured antibodies according to an embodiment of the present invention.
  • Fig. 13 is the result of inhibiting the transformation of hepatic fibroblast LX-2 to fibrosis according to an embodiment of the present invention.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the term "antibody” includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.
  • Whole antibodies are immunoglobulin molecules capable of binding to specific antigens. It includes two light chains with lighter molecular weight and two heavy chains with heavier molecular weight.
  • the heavy chain (H chain) and light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule.
  • the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region), and the carboxy-terminal (C-terminal) is relatively stable, with little change, called the constant region (C region).
  • the V regions of the L and H chains are called VL and VH, respectively.
  • antigen-binding fragment refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • antigen binding moieties include, but are not limited to, variable regions, Fab, Fab", F(ab") 2 , Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2 , multispecific antibodies, Nanobodies.
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked together using recombinant methods through a synthetic linker that enables them to be made into a protein chain in which the VL and VH regions pair to form a monovalent molecule (termed a single-chain Fv (scFv).
  • single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • antigen-binding fragments are obtained using conventional techniques known to those skilled in the art and combined with These fragments are screened for utility in the same way as whole antibodies.
  • Antigen-binding fragments can bind to the same antigen as the parental antibody.
  • antigen-binding fragments may comprise one or more CDRs grafted onto on the framework regions of human antibodies.
  • variable region the amino acid composition and sequence of certain regions have a higher degree of variation, called the hypervariable region (HVR), and the hypervariable region is the position where the antigen and antibody bind, so it is also called the determinant complementarity District (complementarity-determining region, CDR).
  • CDR complementarity-determining region
  • the amino acid composition and sequence of the antibody variable region outside the hypervariable region have relatively small changes, called the framework region (FR).
  • FR framework region
  • the present invention utilizes the IL-11 antigen to obtain a highly specific and high-affinity anti-IL-11 Fab (antigen-binding fragment) antibody fragment through immunization.
  • the antibody fragment can be specifically combined with the IL-11 antigen, so that diseases such as pulmonary fibrosis can be targetedly treated.
  • immunogenicity refers to the ability to cause an immune response, that is, the ability of an antigen to stimulate specific immune cells, activate, proliferate, and differentiate immune cells, and finally produce immune effector substances, antibodies, and sensitized lymphocytes.
  • the inventors of the present application further humanized (modified) the newly screened mouse-derived neutralizing antibody sequence to obtain a humanized antibody, which can retain the characteristics of the parental mouse monoclonal antibody. Affinity and specificity greatly reduce its immunogenicity and improve its safety.
  • the present invention provides a humanized antibody or antigen-binding fragment capable of specifically recognizing IL-11, said antibody containing a CDR sequence selected from at least one of the following or having at least 95% identity therewith Amino acid sequence: heavy chain variable region CDR sequence: SEQ ID NO: 1-33, light chain variable region CDR sequence: SEQ IN NO: 34-66, the antibody or its antigen-binding fragment has humanized modification.
  • the antibodies or antigen-binding fragments provided herein have conservative amino acid substitutions compared to the heavy and light chains described above.
  • Antigen-binding fragment refers to a fragment of an antibody that retains the ability to specifically bind an antigen.
  • Constant amino acid substitution refers to the substitution of an amino acid for another amino acid with a biologically, chemically or structurally similar residue.
  • Biologically similar means that the substitution does not destroy the biological activity of the IL-11 antibody or binding to the IL-11 antigen.
  • Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine, or serine, or have side chains of similar size.
  • Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are substituted for each other, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, and so on.
  • the present invention provides an antibody or antigen-binding fragment
  • the antibody or antigen-binding fragment has a heavy chain variable region of any one of SEQ ID NO: 75 to 78 amino acid sequence and has a sequence such as SEQ ID NO: ID NO: the light chain variable region of the amino acid sequence shown in any one of 79-82.
  • the inventor can obtain the CDR region of the above-mentioned anti-heavy chain variable region sequence (as shown in SEQ ID NO: 4 ⁇ 6) and the CDR region of the light chain variable region sequence (such as Shown in SEQ ID NO:37 ⁇ 39).
  • the heavy chain variable region sequence of the antibody or antigen-binding fragment has conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 75-78.
  • the light chain variable region sequence of the antibody or antigen-binding fragment has conservative amino acid substitutions compared with the amino acid sequence shown in any one of SEQ ID NO:79-82.
  • these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment.
  • these conservative amino acid substitutions may occur at amino acids in the heavy chain variable region and light chain variable region other than the CDR regions.
  • the present invention provides an anti-IL-11 antibody, which has a heavy chain of any one of SEQ ID NO:85-88 amino acid sequence and any one of SEQ ID NO:89-92 The light chain of the amino acid sequence shown.
  • the present invention provides an anti-IL-11 single chain antibody.
  • the nucleic acid molecules expressing the antibodies can be linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.
  • the present invention also provides an isolated nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment.
  • the isolated nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO:93-96 or has a nucleotide sequence shown in any one of SEQ ID NO:97-100.
  • the isolated nucleic acid molecule has at least 90% or more homology with the nucleotide sequence shown in the above-mentioned SEQ ID NO: 93-96, preferably has more than 95% homology, more preferably It has more than 98% and 99% homology.
  • the isolated polynucleotide has at least 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 97-100, preferably more than 95% homology , more preferably having a homology of 98%, 99% or more.
  • sequences have homology with the nucleotide sequence shown in SEQ ID NO:93 ⁇ 96 or SEQ ID NO:97 ⁇ 100, can express and SEQ ID NO:85 ⁇ 88 and SEQ ID NO:89 ⁇ 92 similar amino acid, so that it can specifically bind to the IL-11 antigen and realize the targeting function of the antibody.
  • the isolated nucleic acid molecule has a heavy chain nucleotide sequence shown in SEQ ID NO:93-96 and a light chain nucleotide sequence shown in SEQ ID NO:97-100. These nucleotide sequences are species-optimized for easier expression in mammalian cells.
  • the present invention also provides an expression vector comprising the above-mentioned isolated nucleic acid molecule.
  • the polynucleotide can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • it is sufficient that the polynucleotide is operably linked to the control element.
  • “Operably linked” herein refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function.
  • the polynucleotides used to encode the heavy chain and light chain of the antibody can be independently inserted into different vectors, usually inserted into the same vector.
  • Commonly used vectors can be, for example, plasmids, phages and the like. For example the Plasmid-X plasmid.
  • the invention also provides a recombinant cell containing the expression vector.
  • the expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells can be used to express the antibodies or antigen-binding fragments provided by the present invention.
  • the corresponding antibody can be obtained by culturing the recombinant cells.
  • These usable mammalian cells are, for example, CHO cells and the like.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • anti-IL-11 antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • these pharmaceutical compositions include the anti-IL-11 antibodies provided herein and a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride, are included in the pharmaceutical composition.
  • the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffering agents, to prolong the shelf life or potency of the antibody.
  • antibodies of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms and the like including but not limited to liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • Typical pharmaceutical compositions are in the form of solutions for injection or infusion.
  • the antibodies may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • kits which includes the above-mentioned IL-11 antibody.
  • the application of the kit provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation, and other kits involving the detection of IL-11 antigen and antibody specific binding properties.
  • kits may contain any one or more of the following: antagonists, anti-IL-11 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature wait.
  • Anti-IL-11 antibodies can be used in different types of diagnostic tests, for example, can detect various diseases or the presence of drugs, toxins or other proteins, etc. in vitro or in vivo.
  • the serum or blood of the subject can be tested to test for related diseases.
  • related diseases may include IL-11 related diseases such as pulmonary fibrosis and the like.
  • the antibodies provided herein can also be used for radioimmunoassay and radioimmunotherapy of the above diseases.
  • the anti-IL-11 antibody provided by the present invention can be provided to the subject.
  • the present invention provides a method for treating and/or preventing the above-mentioned diseases, comprising administering the antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, Recombinant cells or pharmaceutical compositions.
  • the amino acid sequence of human IL-11 was obtained through the global public database UniProt (P20809: 124294).
  • the pcDNA3.4 expression vector purchased from Thermo Fisher Scientific) containing the human IL-11 gene sequence was constructed by molecular cloning. Using the method of electroporation, the vector encoding human IL-11 was transfected into mammalian cell CHO for expression for 14 days.
  • the cell supernatant is harvested, and HisTrap HP (nickel column) (GE healthcare, Cat: 17524801) is used for preliminary purification, and then Superdex75 (GE healthcare, Cat: 29148721) is used for fine purification, and finally the obtained protein can be used for immunization Animal recombinant protein antigen hu-IL-11-His tag.
  • HisTrap HP nickel column
  • Superdex75 GE healthcare, Cat: 29148721
  • Embodiment 2 animal immunization
  • mice capable of secreting antibodies against hu-IL-11 the inventors immunized female Balb/c mice.
  • Female Balb/c was purchased from Hunan SJA Laboratory Animal Co., Ltd.
  • the recombinant protein antigen hu-IL-11-His tag obtained in Example 1 and the immune adjuvant were fully emulsified to immunize animals.
  • the immunization process is shown in Table 1.
  • the isolated mouse splenocytes were fused with immortalized mouse myeloma cells SP2/0, and kept in RPMI-1640 complete medium containing 10 -4 M Hypoxanthine, 4x10 -7 M Aminopterin, 1.6x10 -5 M Thymidine After culturing for about 14 days, antibody affinity detection and screening will be carried out after the fused cells grow into clonal clusters. The antibody clones that bind to IL-11 were selected, and their supernatants were taken, and then flow cytometry was performed to determine the competitive binding of antigens and antibodies. Cell lines with high secretion of specific antibodies were selected for expanded culture and cryopreservation. The results are shown in Table 2.
  • the specific method is to firstly obtain an antibody strain with high affinity to IL-11 through Elisa screening (OD450nm is the measured affinity value), wherein PC is the difference between the purchased IL-11 antibody (Abcam, ab130083) and IL-11 protein The combination was used as a positive control.
  • N/A in Table 2 means: no data
  • N/A in Table 3 means: no data; because the sequence of 3-6A4 cells after subcloning is consistent with the sequence before subcloning, its number is the same as that in Table 2.
  • the design of primers for gene amplification of antibody heavy chain variable region (VH) and light chain variable region (VL) mainly refers to the primer pair in Ig-Primer Sets (Novagen Company, catalog number: 69831-3), synthesized by BGI .
  • the heavy chain variable region and light chain variable region genes were amplified from the first strand of cDNA, cloned into the pMD18-T vector, transformed into Escherichia coli DH5 ⁇ , and the clones were picked and sequenced by BGI to obtain the heavy chain variable region (VH) and light chain variable region (VL) gene sequences.
  • the sequence of the antibody obtained by sequencing is shown in the following SEQ ID NO: 101-122.
  • the antibody composed of the above SEQ ID NO: 101 and 112 is called 3-6A4, the antibody composed of the above SEQ ID NO: 102 and 113 is called 5-20E11-5F2, and the antibody composed of the above SEQ ID NO: 103 and 114
  • the antibody is called 3-5B2-2G4, the antibody consisting of the above SEQ ID NO: 104 and 115 is called 3-5B2-7A4, and the antibody consisting of the above SEQ ID NO: 105 and 116 is called 3-2B5 -3D2,
  • the antibody composed of the above SEQ ID NO:106 and SEQ ID NO:117 is called 3-7C9-1A6, and the antibody composed of the above SEQ ID NO:107 and SEQ ID NO:118 is called 3-9E1 -3A2
  • the antibody composed of the above SEQ ID NO:108 and SEQ ID NO:119 is called 3-15D5-3A6, and the antibody composed of the above SEQ ID NO:109 and SEQ ID NO:120 is called 3-15D5 -6A1, the antibody composed of the
  • the monoclonal hybridoma cell line corresponding to the preferred antibody strain was injected into Balb/C mice by conventional methods to produce and purify the ascites antibody.
  • the purified antibody was used for antibody subtype analysis, and the antibody subtype detection method was detected by a mature kit (Proteintech, KMIM-2). The results are shown in Table 4 below.
  • the inventors constructed the sequences of the preferred monoclonal antibody strains 3-6A4 and 5-20E11-5F2 into conventional expression vectors by genetic engineering, and then expressed them in CHO cells. After the expression was completed, the collected cell culture medium was purified by Protein G chromatography column (EzFast Protein G 4FF, Borglon), the balance liquid was 20mM PBS, 0.15M NaCl, pH7.4, and the eluent was 0.1M glycine pH3 .0 ⁇ 0.2, collect the protein eluate under the target absorption peak, after ultrafiltration with PBS buffer, take some samples for SDS-PAGE electrophoresis detection, and use SEC-HPLC method to determine the polymer content. The multimer content of the antibody is all within 3%.
  • the inventors coated an appropriate amount of IL-11R-His protein on an Elisa detection plate and incubated overnight. Then by adding an appropriate amount of IL-11-Fc protein, and co-incubating with antibodies of different concentration gradients. Finally, HRP-coupled Goat anti IgG-Fc secondary antibody (ThermoFisher, 31413) and TMB chromogenic substrate were used for detection. The inventors detected that the preferred antibody and 6D9A1 (Abcam, ab130083) could inhibit the binding of IL-11 and IL-11R to varying degrees, and the results are shown in FIG. 1 .
  • IL-6 cytokine family members include IL-6, IL-11, CNTF, CLC, LIF, CT-1, OSM, IL-27, IL-31.
  • IL-11 is a member of the IL-6 cytokine family.
  • the inventors used the conventional Elisa method to coat recombinant IL-6 (Sinobiological-10395-HNAE), IL-27 (R&D systems, 2526-IL-010), LIF (Peprotech, 300-05), CLC (R&D systems, 962- CL-050), IL-11 (LSBio, LS-G132887-10), OSM (Genscript-z03132), IL-31 (Peprotech, 200-31), CNTF (R&D systems, 257-NT-010), CT- 1 (R&D systems, 612-CD-010) protein, and the binding of preferred antibodies 3-6A4 and 5-20E11-5F2 to IL-6 cytokine family members was detected. The results show that the
  • Biofilm interferometry was used to measure, and Ni-NTA sensor was used to capture antigen IL-11 (His tag).
  • Antibody strains 3-6A4 and 5-20E11-5F2 were preferably used as analytes, and the analytes were diluted in 7 concentration gradients. The time is 60s, and the dissociation time is 1000s.
  • the buffer is 20mM PBS, 0.15M NaCl, 0.1% BSA, 0.05% Tween 20, pH7.4.
  • the inventors constructed stable expression full-length IL-11R
  • the cell line membrane-IL-11R CHO was used to detect the blocking activity of candidate antibody and commercial antibody 6D9A1 (Abcam, ab130083) on IL-11 and IL-11R by flow cytometry. It is preferred to incubate an appropriate amount of IL-11 recombinant protein with Fc tag with different gradient antibodies for 15mins, and then incubate with 1E+05 membrane-IL-11R CHO cells.
  • the secondary antibody used in the detection was a secondary antibody coupled with a FITC fluorophore (Goat-anti-Fc-FITC) (Abcam, ab98529).
  • the competitive binding results showed that the neutralizing activity IC50 of the 3-6A4 antibody was about 7 ⁇ g/mL;5 -
  • the neutralizing activity IC50 of the 20E11-5F2 antibody is about 21 ⁇ g/mL, and the blocking activity of the candidate antibody is significantly better than that of the 6D9A1 clone antibody.
  • Figure 2 The results are shown in Figure 2.
  • Example 11 Antibody strains inhibit the transformation of lung fibroblast HFL-1 to fibrosis
  • HFL-1 cells as lung fibroblasts, will express a large amount of IL-11 cytokines when induced by pro-fibrotic factors such as TGF-beta1 (TGF ⁇ ), and eventually transform into fibrosis.
  • TGF-beta1 TGF-beta1
  • the marker ACTA2 protein will continue to accumulate.
  • the inventors used TGF-beta1 cytokines to induce the transformation of HFL-1 cells to fibrosis while adding the preferred 3-6-A4 and 5-20E11-5F2 antibody strains.
  • the preferred antibodies of different concentrations can inhibit the transformation of HFL-1 cells to fibrosis (the expression of ACTA2 protein decreases ), as shown in Figure 3 and the corresponding statistical analysis Figure 4.
  • Example 12 Antibodies inhibit pulmonary fibrosis in C57BL/6 mice
  • the humanization of the IL-11 antibody in the above example was performed by CDR grafting.
  • the germline selected for the humanization of the antibody heavy chain variable region is IGHV1-46*01.
  • IGKV3-11*01 was selected for humanization of the chain variable region.
  • the CDR region of the murine antibody is retained, and the framework sequence of the murine antibody is replaced with the framework sequence of a human germline antibody.
  • the mouse antibody Fv fragment structure model was established, BLAST was performed in the PDB antibody database, and the structure INQB was selected as a template for homology modeling.
  • the sequences of the heavy chain variable region and the light chain variable region of the obtained IL-11 humanized antibody are shown in SEQ ID NO: 75-78 and SEQ ID NO: 79-82.
  • VH1 humanized antibody heavy chain variable region sequence
  • VH2 IL-11 humanized antibody heavy chain variable region sequence
  • VH3 IL-11 humanized antibody heavy chain variable region sequence
  • VH4 IL-11 humanized antibody heavy chain variable region sequence
  • VL1 IL-11 humanized antibody light chain variable region sequence
  • VL2 IL-11 humanized antibody light chain variable region sequence
  • VL3 IL-11 humanized antibody light chain variable region sequence
  • VL4 IL-11 humanized antibody light chain variable region sequence
  • nucleic acid sequences encoding the light chain and heavy chain of IL-11 humanized antibody were synthesized and inserted into the eukaryotic expression vector pcDNA3.4, and the vectors of the four heavy chain sequences were paired with the vectors of the four light chain sequences respectively, Co-transfected into mammalian cell CHO for expression to obtain scFv single-chain antibody. One week later, the supernatant containing the scFv antibody was collected for affinity detection.
  • Biacore was used to measure the binding of the supernatant scFv of each humanized single-chain antibody obtained in Example 13 to IL-11, wherein the connecting peptide in the humanized single-chain antibody was GGGGSGGGGSGGGGS (SEQ ID NO: 123), According to the dissociation constant kd (1/s), the three humanized antibodies with the smallest dissociation constant kd (1/s) were selected.
  • the assay method includes the following steps: capturing the humanized single-chain antibody at a flow rate of 10 ⁇ L/min. Switch the flow rate to 30 ⁇ L/min, flow 400 nM IL-11 antigen through the sample channel, the binding time is 180 s, and the dissociation time is 600 s.
  • the preferred IL-11 humanized antibodies "VH1+VL2", “VH1+VL3", “VH1+VL4", and 5-20E11-5F2 were respectively constructed for full-length sequences (containing the Fc of mouse-derived wild-type IgG1 fragment), obtain the pxC17.4&pxC18.4 expression vector containing the full length of the antibody and transfect it into mammalian cell CHO for expression.
  • the collected cell culture fluid is purified by Protein A chromatography column, the balance fluid is 20mM PBS, pH7.4, the eluent is 0.1M glycine pH3.0 ⁇ 0.2, and the protein eluate under the target absorption peak is collected After ultrafiltration with PBS buffer, some samples were taken to measure the concentration by HPLC and SDS-PAGE electrophoresis detection.
  • SPR surface plasmon resonance
  • Biacore T200 GE Healthcare
  • the assay method comprises the following steps: capturing the full-length humanized antibody at a flow rate of 10 ⁇ L/min. Switch the flow rate to 30 ⁇ L/min, and different concentrations of IL-11 antigen (400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM) flow through the sample channel and the reference channel, the binding time is 180s, and the dissociation time is 600s. Finally the chip was regenerated with 10 mM glycine buffer.
  • the affinity of the full-length humanized antibody constructed by "VH1+VL3" is better than that of the other two full-length humanized antibodies, and the affinity is slightly lower than that of the full-length chimeric antibody constructed by 5-20E11-5F2 There is a reduction.
  • VH1+VL2 "VH1+VL3" and “VH1+VL4" in Table 8 are all humanized full-length (IgG form) antibodies.
  • the heavy chain of the full-length humanized antibody sequence of "VH1+VL2" is SEQ ID NO:85, and the light chain is SEQ ID NO:90;
  • the heavy chain of the full-length humanized antibody sequence of "VH1+VL3" is SEQ ID NO: :85, the light chain is SEQ ID NO:91;
  • the heavy chain of the "VH1+VL4" full-length humanized antibody sequence is SEQ ID NO:85, and the light chain is SEQ ID NO:92.
  • Example 15 The thermal stability of each of the full-length humanized or chimeric antibodies obtained in Example 15 was analyzed and determined by MicroCal TM VP-Capillary DSC system.
  • the antibody was diluted to 0.4mg/mL with PBS buffer, and the measurement conditions were: heating rate 90°C/h, temperature range 25-110°C.
  • the results are shown in Table 9 and Figure 7. Compared with 5-20E11-5F2, the Tm value of the humanized antibody "VH1+VL3" was increased by 4.5°C.
  • VH1+VL2 "VH1+VL3" and “VH1+VL4" in Table 9 and Figure 7 are all humanized full-length (IgG format) antibodies; in Figure 7, “VH+VL” is 5-20E11- 5F2 full-length antibody.
  • Example 18 Preferred Humanized Antibody Competitively Blocks the Combination of IL-11 and IL-11R
  • the inventors constructed a cell line membrane-IL-11R CHO stably expressing IL-11R.
  • the assay method includes the following steps: 10 ⁇ L/well of IL-11-Fc protein with a concentration of 100 ⁇ g/mL is added to a 96-well U-shaped plate, and 90 ⁇ L of the full-length chimeric antibody 5-20E11 obtained in Example 15 is serially diluted -5F2 and "VH1+VL2", "VH1+VL3" and "VH1+VL4" constructed full-length humanized antibodies (100 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL, 0.1 ⁇ g/mL, 0.01 ⁇ g/mL mL, 0.001 ⁇ g/mL) and incubated at room temperature for 1 h.
  • VH1+VL2 "VH1+VL3" and “VH1+VL4" in Table 10 and Figure 8 are all humanized full-length (IgG) antibodies.
  • LX-2 cells are human hepatic stellate cells. When induced by pro-fibrotic factors such as TGF-beta1 (TGF- ⁇ ), they will express a large amount of IL-11 cytokines and eventually transform into fibrosis. During the transformation of LX-2 cells to fibrosis, the marker ACTA2 protein will continue to accumulate.
  • TGF- ⁇ pro-fibrotic factors
  • the marker ACTA2 protein will continue to accumulate.
  • the inventors spread LX-2 cells on a 6-well plate at a density of 3*10 ⁇ 5 cells/well and cultured them for 10 hours until the cells adhered to the wall.
  • the full-length chimeric antibody (CPD-1) and "VH1+VL3" constructed by the chemical medicine (HEC585 (iporphinone hydrochloride), BIBF1120) and the full-length chimeric antibody (CPD-1) obtained in Example 15 obtained from 5-20E11-5F2
  • the long humanized antibody (CPD-2, ie the "VH1+VL3" mAb) was subjected to animal experiments, wherein an IgG antibody was used as a control.
  • the specific experimental operation is as follows:
  • the rats were anesthetized with 2.5% isoflurane, and after the main trachea was opened, an appropriate amount of vehicle or bleomycin was injected to establish the model, wherein, bleomycin (Bleomycin) was used to induce the construction of SD rat unilateral lung As an animal model of fibrosis, the recovery of rats was observed after surgery. On the 8th day after the operation, intragastric administration or intraperitoneal administration was carried out for 14 days of treatment. The specific animal groups and administration doses are summarized in Table 11 below. After humanized antibody treatment, it can be clearly observed that the humanized antibody slows down the inflammatory response and fibrosis process of rat lung.
  • VH1+VL3 in Table 11 is a humanized full-length (IgG form) antibody.
  • HE staining mainly indicates the infiltration level of inflammation in lung tissue.
  • Masson's trichrome staining is mainly used to evaluate the level of lung tissue fibrosis.
  • Results Figure 9 and Figure 10 both show that the preferred humanized antibody can well reduce the pneumonia damage and fibrosis in rats induced by bleomycin.
  • the effect of humanized antibody on pneumonia injury in rats was significantly better than that of 5-20E11-5F2 antibody.
  • VH1+VH3 was constructed into a ScFv in the form of VH-Linker-VL, which was used as a template for phage library construction and multiple rounds of panning. After ELISA and flow detection, candidates with higher affinity than the original sequence were selected thing.
  • the CDR mutation library and the random mutation library were constructed, and the two libraries were constructed separately, and the phages were mixed during panning.
  • Antibody panning use different concentrations of IL-11 antigen for the CDR mutation library and random mutation library, perform multiple rounds of liquid phase screening for phages that can bind IL-11, and detect their affinity by phage ELISA and flow cytometry characteristics, and then sequenced to obtain a total of 14 clones with better affinity activity. The results of flow cytometry and sequencing are shown in Table 12.
  • the assay method includes the steps of capturing the humanized antibody with an AHC sensor.
  • the analytes were different concentrations of IL-11 antigen (125nM, 62.5nM, 31.3nM, 15.6nM, 7.81nM, 3.91nM), the binding time was 200s, and the dissociation time was 1000s.
  • the sensor was regenerated with 10 mM glycine (pH 1.7) buffer.
  • the affinity of the antibodies MIL-11mab-02 and MIL-11mab-03 after affinity maturation is 10 times higher than that of the humanized antibodies, and the full-length human origins constructed with "VH1+VH3" and wild-type IgG1Fc fragments Antibodies have comparable affinity.
  • VH1+VL3 in Table 13 is a humanized full-length (IgG form) antibody.
  • the inventors constructed a cell line membrane-IL-11CHO stably expressing IL-11. Take the affinity maturation antibody obtained in Example 21 (100 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL, 0.1 ⁇ g/mL, 0.01 ⁇ g/mL, 0.001 ⁇ g/mL) and 1.5*10 ⁇ 5 membrane-IL -11CHO cells were incubated at room temperature for 1 hour, washed 3 times with PBS, added flow cytometric secondary antibody Goat-anti-Fc-FITC (Abcam, ab98529), and detected by flow cytometry. The resulting S-curve is shown in Figure 11.
  • Hu-IL11mab refers to the humanized antibody constructed from "VH1+VH3". The results show that the binding activity of the affinity matured antibody is basically the same as that of the humanized antibody.
  • the assay method comprises the following steps: 10 ⁇ L/well of IL-11-His protein with a concentration of 10 ⁇ g/mL is added to a 96-well U-shaped plate, and 90 ⁇ L of gradiently diluted preferred humanized antibodies (100 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g /mL, 0.1 ⁇ g/mL, 0.01 ⁇ g/mL, 0.001 ⁇ g/mL) at room temperature for 1h.

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Abstract

提供了一种能够特异性识别IL-11的抗体或其抗原结合片段。该抗体包括分别如SEQ ID NO:4-6所示的重链可变区CDR，分别如SEQ ID NO:37-39所示的轻链可变区CDR，分别如SEQ ID NO:67-70所示的重链框架区，和分别如SEQ ID NO:71-74所示的轻链框架区。该抗体免疫原性低，能够特异性的靶向结合IL-11，阻断IL-11和IL-11受体的结合。

Description

IL-11人源化抗体及其应用 技术领域
本发明涉及生物技术领域，具体地，本发明涉及IL-11的抗体及其应用，更具体地，本发明涉及能够特异性识别IL-11的抗体或其抗原结合片段、核酸分子、表达载体、重组细胞、药物组合物、制药用途、检测IL-11的试剂盒以及制备检测或诊断试剂盒的用途。
背景技术
纤维化是一个重要的过程，是伤口愈合的关键部分。过度纤维化在许多罕见和常见疾病中很常见，并且在疾病发病机制中很重要。以过度纤维化为特征的疾病包括但不限于：系统性硬化症、硬皮病、肥厚性心肌病、扩张型心肌病(DCM)、心房纤维性颤动、心室纤维性颤动、心肌炎、肝硬化、肾脏疾病、眼部疾病、哮喘、囊性纤维化、关节炎和特发性肺纤维化。尽管对人类健康影响很大，但对纤维化的治疗和诊断方法的医疗需求仍然未得到满足。
白细胞介素11(IL-11)的真正生理作用尚不清楚。IL-11与造血细胞活化和血小板生成的关系最为密切，但也被发现具有促炎和抗炎、促血管生成的作用，并对瘤形成很重要。已知TGFβ1或组织损伤可诱导IL-11的表达。从已发表的文献来看，IL-11在纤维化中的作用尚不清楚。IL-11被认为对肺纤维化和炎症很重要，其表达水平与皮肤和呼吸系统中的胶原水平相关。然而，大多数研究表明IL-11在心脏和肾脏中是抗纤维化的，在几种组织和慢性炎症疾病中的是抗炎的。一般来说，IL-11的分子作用模式被认为是通过STAT3介导的转录调节RNA表达的mRNA水平。
能够抑制IL-11作用的试剂可以阻止或减少IL-11与IL-11受体的结合。已有研究表明，施用能够抑制白细胞介素11(IL-11)作用的试剂来治疗、预防或缓解需要治疗的受试者中的纤维化。因此，研究能够有效结合IL-11的抗体对于纤维化疾病的治疗具有极大价值。
发明内容
本发明开发一款具有高中和活性人源化的Anti-IL-11的抗体，以期用于纤维化相关疾病的治疗或预防。
在本发明的的第一方面，本发明提出了一种能够特异性识别IL-11的抗体或其抗原结合片段。根据本发明的实施例，所述抗体含有选自下列至少之一的CDR序列或与其具有至少95％同一性的氨基酸序列：重链可变区CDR序列：SEQ ID NO:1～33，轻链可变区CDR序列：SEQ IN NO:34～66，所述抗体或其抗原结合片段具有人源化修饰。需要说明的是，本申请所述的“人源化修饰”是指能够使得所述抗体或其抗原结合片段的免疫原性降低的氨基酸的改变，包括氨基酸的突变、插入、缺失、化学基团的赘合等等。
Figure PCTCN2022111608-appb-000001
Figure PCTCN2022111608-appb-000002
根据本发明实施例的上述人源化抗体的免疫源性降低，抗体能够特异性的靶向结合IL-11，阻断IL-11和IL-11受体的结合。
根据本发明的实施例，上述抗体或抗原结合片段还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述抗体包括：分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
根据本发明的实施例，所述抗体包括：分别如SEQ ID NO:37、38和39或者与SEQ ID NO:37、38和39具有至少95％同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。
根据本发明的实施例，所述抗体或其抗原结合片段特异性识别IL-11。
根据本发明的实施例，所述抗体含有重链框架区序列和轻链框架区序列的至少之一，所述重链框架区序列和轻链框架区序列的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
根据本发明的实施例，所述重链框架区包括FL1、FL2、FL3和FL4，所述重链框架区FL1、FL2、FL3和FL4分别具有SEQ ID NO:67、68、69和70所示的氨基酸序列，
Figure PCTCN2022111608-appb-000003
其中，X1为Q或H,X2为M或I，X3为V或A，X4为M或L，X5为R或A，X6为Y或F。
根据本发明的实施例，所述轻链框架区包括FL1、FL2、FL3和FL4，所述轻链框架区FL1、FL2、FL3和FL4分别具有SEQ ID NO:71、72、73和74所示的氨基酸序列，
Figure PCTCN2022111608-appb-000004
其中，X7为Y或F,X8为V或M，X9为Y或F。
根据本发明的实施例，所述抗体具有如SEQ ID NO：75～78任一项所示氨基酸序列的重链可变区。
Figure PCTCN2022111608-appb-000005
Figure PCTCN2022111608-appb-000006
根据本发明的实施例，所述抗体具有如SEQ ID NO：79～82任一项所示氨基酸序列的轻链可变区。
Figure PCTCN2022111608-appb-000007
根据本发明的实施例，所述抗体含有重链恒定区和轻链恒定区的至少之一，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
根据本发明的实施例，所述抗体恒定区的全长序列如SEQ ID NO:83或84所示。
Figure PCTCN2022111608-appb-000008
其中，上述SEQ ID NO:83所示的抗体恒定区的全长序列包括IgG1重链恒定区和Fc区，其中，IgG1重链恒定区序列为ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV，Fc区序列为EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；上述SEQ ID NO:84所示的抗体恒定区的全长序列为IgG轻链恒定区。
根据本发明的实施例，所述抗体具有SEQ ID NO:85～88任一项所示氨基酸序列的重链和具有SEQ ID NO:89～92任一项所示氨基酸序列的轻链。
Figure PCTCN2022111608-appb-000009
Figure PCTCN2022111608-appb-000010
根据本发明的实施例，所述抗体为单链抗体、多聚体抗体、CDR移植抗体或小分子抗体。
根据本发明的实施例，所述小分子抗体包括Fab抗体、Fv抗体、单域抗体以及最小识别单位的至少之一。
在本发明的第二方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码前面所述的抗体或其抗原结合片段。根据本发明实施例的核酸分子所编码的抗体或抗原结合片段免疫源性低，可特异性靶向结合IL-11，阻断IL-11和IL-11受体的结合。
根据本发明的实施例，上述核酸分子还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述核酸分子为DNA。
根据本发明的实施例，所述核酸分子具有如SEQ ID NO:93～96任一项所示核苷酸序列或具有SEQ ID NO:97～100任一项所示核苷酸序列。
Figure PCTCN2022111608-appb-000011
Figure PCTCN2022111608-appb-000012
Figure PCTCN2022111608-appb-000013
Figure PCTCN2022111608-appb-000014
Figure PCTCN2022111608-appb-000015
在本发明的第三方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带前面所述的核酸分子。根据本发明实施例的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的特异性识别IL-11的抗体或其抗原结合片段表达，进而实现所述抗体或抗原结合片段的体外大量获得。
根据本发明的实施例，上述表达载体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述表达载体为真核表达载体。进而实现前面所述的特异性识别IL-11的抗体或其抗原结合片段在真核细胞中的表达，如CHO细胞。
在本发明的第四方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带前面所述的核酸分子，或者表达前面所述的抗体或其抗原结合片段。根据本发明实施例的重组细胞可用于前面所述的特异性识别IL-11的抗体或其抗原结合片段体外表达和大量获得。
根据本发明的实施例，上述重组细胞还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述重组细胞是通过将前面所述的表达载体引入至宿主细胞中而获得的。
根据本发明的实施例，通过电转导的方法将所述表达载体引入所述宿主细胞中。
根据本发明的实施例，所述重组细胞为真核细胞。
根据本发明的实施例，所述重组细胞为哺乳动物细胞。
在本发明的第五方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物含有前面所述的抗体，前面所述的核酸分子，前面所述的表达载体或前面所述的重组细胞。根据本发明实施例的药物组合物中所包含的抗体或表达的抗体能够特异性的靶向结合IL-11，阻断IL-11和IL-11受体的结合。
在本发明的第六方面，本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体或前面所述的重组细胞、前面所述的药物组合物在制备药物中的用途，所述药物用于治疗或者肺纤维化(Pulmonary fibrosis)，非酒精性脂肪肝炎(non-alcoholic steatohepatitis,NASH)，慢性心力衰竭(Chronic heart failure,CHF)或由成纤维细胞向纤维化转化所导致的疾病。
在本发明的第七方面，本发明提出了一种检测IL-11的试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的抗体。前面所述的IL-11抗体能够特异性靶向结合IL-11，根据本发明实施例的试剂盒可以实现IL-11的特异性检测，如当抗体结合有荧光基团时，可以采用荧光检测装置实现对IL-11的定位或实时检测。
在本发明的第八方面，本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体或前面所述的重组细胞在制备试剂盒中的用途，所述试剂盒用于检测IL-11或者诊断IL-11相关的疾病。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的优选抗体竞争性阻断IL-11与IL-11R结合的结果；
图2是根据本发明实施例的优选抗体竞争性阻断活性检测结果；
图3是根据本发明实施例的优选抗体株抑制肺成纤维细胞HFL-1向纤维化转化的western检测结果；
图4是根据本发明实施例的优选抗体株抑制肺成纤维细胞HFL-1向纤维化转化统计分析结果；
图5和图6是根据本发明实施例的优选抗体在C57BL/6小鼠中抑制肺纤维化结果。
图7是根据本发明实施例的优选人源化抗体热稳定性检测结果图；
图8是根据本发明实施例的人源化抗体竞争性阻断活性；
图9是根据本发明实施例的人源化抗体治疗后SD大鼠肺部炎症损伤评分；
图10是根据本发明实施例的人源化抗体治疗SD大鼠肺部纤维化评分；
图11是根据本发明实施例的亲和力成熟抗体结合活性；
图12是根据本发明实施例的亲和力成熟抗体竞争阻断活性；以及
图13是根据本发明实施例的抑制肝成纤维细胞LX-2向纤维化转化结果。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指 示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
抗体
本文中，术语“抗体”包括完整抗体及其任何抗原结合片段(即，“抗原结合部分”)或单链。完整抗体是能够与特异性抗原结合的免疫球蛋白分子。包括两条分子量较轻的轻链和两条分子量较重的重链，重链(H链)和轻链(L链)由二硫键连接形成一个四肽链分子。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)，羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。L链和H链的V区分别称为VL和VH。
术语“抗原结合片段”(或简称“抗体片段”)是指由包含一个或多个CDR的抗体的一部分形成的抗体片段。已显示抗体的抗原结合功能可由全长抗体的片段来行使。抗原结合部分的例子包括但不限于，可变区、Fab、Fab”、F(ab”) 2、Fv片段、二硫化键稳定的Fv片段(dsFv)、(dsFv) 2、多特异性抗体、纳米抗体(nanobody)。此外，尽管Fv片段的两个结构域VL和VH由单独的基因编码，但是它们可以利用重组方法通过合成连接体连接在一起，该连接体使它们能够制成一条蛋白质链，其中VL和VH区配对形成单价分子(称为单链Fv(scFv)。这种单链抗体也预期包括在术语抗体的“抗原结合片段”内。这些抗体片段用本领域技术人员已知的常规技术获得，并用与完整抗体相同的方法对这些片段的实用性进行筛选。抗原结合片段能与亲本抗体结合相同的抗原。在一些实施方案中，抗原结合片段可包含一个或多个CDR嫁接到来自一个或多个不同人源抗体的框架区上。
在可变区中某些区域氨基酸组成和排列顺序具有更高的变化程度，称为高变区(Hypervariable region，HVR)，高变区为抗原和抗体结合的位置，因此也称为决定簇互补区(complementarity-determining region，CDR)。重链可变区和轻链可变区上均有三个CDR区。抗体可变区内高变区之外的氨基酸组成和排列顺序变化相对较小，称为框架区(FR)。VH和VL内各有4个框架区，分别以FR1、FR2、FR3和FR4表示。
本发明利用IL-11抗原，通过免疫获得了高特异性的高亲和力的抗IL-11的Fab(antigen-binding fragment)抗体片段。利用该抗体片段能够与IL-11抗原特异性结合，从而可以靶向性治疗肺纤维化等疾病。
需要说明的是，“免疫原性”是指能引起免疫应答的性能，即抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质抗体和致敏淋巴细胞的特性。本申请的发明人进一步对筛选得到的鼠源的全新中和抗体序列进行人源化改造(人源化修饰)，得到人源化抗体，该人源化抗体既能保留亲本鼠单克隆抗体的亲和力和特异性，又大幅降低了其免疫原性，提高了其安全性。
在一些实施方案中，本发明提供了一种能够特异性识别IL-11的人源化抗体或者抗原结合片段，所述抗体含有选自下列至少之一的CDR序列或与其具有至少95％同一性的氨基酸序列：重链可变区CDR序列：SEQ ID NO:1～33，轻链可变区CDR序列：SEQ IN NO:34～66，所述抗体或其抗原结合片段具有人源化修饰。在另一些实施方案中，本发明所提供的抗体或者抗原结合片段与上述重链和轻链相比，具有保守氨基酸取代。“抗原结合片段”是指保持特异性结合抗原能力的抗体片段。“保守氨基酸取代”指的是氨基酸被另一氨基酸发生生物学上、化学上或者结构上相似的残基所取代。生物学上相似的指的是该取代不破坏 IL-11抗体或者与IL-11抗原结合的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链，如丙氨酸、甘氨酸或丝氨酸，或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者极性氨基酸相互取代，例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬酰胺，丝氨酸取代苏氨酸等等。
在一些实施方案中，本发明提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段具有SEQ ID NO：75～78任一项所示氨基酸序列的重链可变区和具有如SEQ ID NO：79～82任一项所示氨基酸序列的轻链可变区。发明人通过抗体序列比对数据库(NCBI、IMGT)可得到上述抗重链可变区序列的CDR区(如SEQ ID NO:4～6所示)和轻链可变区序列的CDR区(如SEQ ID NO:37～39所示)。在另一些实施方案中，所述抗体或抗原结合片段的重链可变区序列与SEQ ID NO:75～78所示氨基酸序列相比，具有保守氨基酸取代。在一些实施方案中，所述抗体或抗原结合片段的轻链可变区序列与SEQ ID NO:79～82任一项所示氨基酸序列相比，具有保守氨基酸取代。当然，这些保守氨基酸取代不会对抗体或者抗原结合片段的生物学功能带来改变。在一些具体方式中，这些保守氨基酸取代可以发生在重链可变区和轻链可变区中除了CDR区之外的氨基酸上。
在一些优选方案中，本发明提供了一种抗IL-11抗体，该抗体具有SEQ ID NO:85～88任一项所示氨基酸序列的重链和具有SEQ ID NO:89～92任一项所示氨基酸序列的轻链。
在一些优选方案中，本发明提供了一种抗IL-11单链抗体。
核酸分子、表达载体、重组细胞
在制备或者获取这些抗体的过程中，可以利用表达所述抗体的核酸分子，与不同的载体连接，然后在不同细胞中表达，来获得相应抗体。
为此，本发明还提供了一种分离的核酸分子，所述核酸分子编码上述所述的抗体或抗原结合片段。
在一些实施方案中，所述分离核酸分子具有如SEQ ID NO:93～96任一项所示核苷酸序列或具有SEQ ID NO:97～100任一项所示核苷酸序列。
在一些实施方案中，所述分离的核酸分子与上述SEQ ID NO:93～96所示的核苷酸序列至少具有90％以上的同源性，优选具有95％以上的同源性，更优选具有98％、99％以上的同源性。在至少一些实施方案中，所述分离的多核苷酸与所述SEQ ID NO:97～100所示的核苷酸序列至少具有90％以上的同源性，优选具有95％以上的同源性，更优选具有98％、99％以上的同源性。这些与SEQ ID NO:93～96或者SEQ ID NO:97～100所示核苷酸序列具有同源性的序列，能够表达与SEQ ID NO:85～88和SEQ ID NO:89～92相似的氨基酸，从而能够与IL-11抗原特异性结合，实现抗体的靶向性功能。
在一些优选实施方式中，所述分离的核酸分子具有SEQ ID NO:93～96所示的重链核苷酸序列和SEQ ID NO:97～100所示的轻链核苷酸序列。这些核苷酸序列经过种属优化，更易在哺乳动物细胞中表达。
本发明还提供了一种表达载体，所述表达载体包含上述分离的核酸分子。在将上述分离的多核苷酸连接到载体上时，可以将多核苷酸与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制多核苷酸的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自 于载体本身。当然，多核苷酸与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码抗体重链和轻链的多核苷酸，可以分别独立的插入到不同的载体上，常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。例如Plasmid-X质粒。
本发明还提供了一种重组细胞，该重组细胞中包含有该表达载体。可以将表达载体导入到哺乳动物细胞中，构建获得重组细胞，然后利用这些重组细胞表达本发明提供的抗体或者抗原结合片段。通过该重组细胞进行培养，即可以获得相应抗体。这些可用的哺乳动物细胞例如可以为CHO细胞等。
药物组合物、试剂盒及制药用途和在制备试剂盒中的用途。
本发明还提供了一种药物组合物，所述药物组合物包括上述抗体或者抗原结合片段和药学可接受的载体。
本文提供的抗IL-11抗体可以掺入适合受试者施用的药物组合物中。通常，这些药物组合物包括本文提供的抗IL-11抗体以及药学上可接受的载体。
“药学上可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等等。具体实例可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等以及它们的组合物中的一种或多种。有许多情况下，药物组合物中包括等渗剂，例如糖类、多元醇(如甘露醇、山梨醇)或氯化钠等。当然药学上可接受的载体还可包括微量的辅助物质，例如润湿剂或乳化剂、防腐剂或缓冲剂，用来延长抗体的保存限期或效力。
例如，本发明的抗体可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物组合物为注射溶液或输注溶液形式。所述抗体可通过静脉输注或注射或肌肉内或皮下注射来施用。
当然，本文中的抗IL-11抗体还可以根据需要被制成试剂盒或者其他诊断性试剂的一部分。根据本发明的实施例，本发明还提供了一种试剂盒，所述试剂盒包括上述IL-11抗体。应用本发明提供的试剂盒，例如可以用于免疫印迹、免疫沉淀等涉及到利用IL-11抗原和抗体特异性结合性能，来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、抗IL-11抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。抗IL-11抗体可被用于不同类型的诊断测试，例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在。例如可以通过对受试者的血清或者血液进行检测，用来测试相关疾病。这种相关疾病可包括IL-11相关疾病，例如肺纤维化等等。当然本文提供的抗体也可以用于上述疾病的放射免疫检测和放射免疫治疗等等。
在利用本发明所提供的抗IL-11抗体治疗上述疾病时，可以将本发明提供的抗IL-11抗体提供给受试者即可。为此，本发明提供了一种用于治疗和/或预防上述疾病的方法，包括向有需要的受试者施用本发明所提供的所述抗体或其抗原结合片段、核酸分子、表达载体、重组细胞或药物组合物。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 重组人源蛋白抗原hu-IL-11-His tag的生产
载体的构建：人源IL-11氨基酸序列通过全球公共数据库UniProt(P20809：124294)获得。采用分子克隆的方法，构建了含人源IL-11基因序列的pcDNA3.4,表达载体(购自Thermo Fisher Scientific)。采用电转染的方法，将编码人源IL-11的载体转染至哺乳动物细胞CHO进行表达14天。待表达完成收获细胞上清液，采用HisTrap HP(镍柱)(GE healthcare,Cat:17524801)进行初步纯化，然后再采用Superdex75(GE healthcare,Cat:29148721)进行精细纯化，最终获得可以用于免疫动物的重组蛋白抗原hu-IL-11-His tag。
实施例2 动物免疫
为了得到能分泌针对hu-IL-11抗体的小鼠，发明人对雌性Balb/c小鼠进行免疫。雌性Balb/c购买于湖南斯莱克景达实验动物有限公司(Hunan SJA Laboratory Animal Co.，Ltd)。将实施例1获得的所述重组蛋白抗原hu-IL-11-His tag与免疫佐剂经充分乳化后进行动物免疫，免疫流程见表1。
表1：免疫动物安排表
Figure PCTCN2022111608-appb-000016
第35天收获少量的小鼠眼眶血，采用常规的Elisa方法进行抗体效价检测。
实施例3 抗体分泌细胞融合与筛选
分离的小鼠脾细胞与永生化的小鼠骨髓瘤细胞SP2/0融合，并使用含10 -4M Hypoxanthine,4x10 -7M Aminopterin,1.6x10 -5M Thymidine的RPMI-1640完全培养基静置培养约14天，待融合细胞长出克隆团后进行抗体亲和力的检测筛选。选取与IL-11结合的抗体克隆株，取其上清，再进行流式细胞术测定抗原抗 体的竞争结合，选出高分泌特异性抗体细胞株进行扩大培养及冻存，结果见表2。具体方法是首先通过Elisa筛选获得与IL-11具有高亲和力的抗体株(OD450nm即为测定的亲和力值)，其中P.C.为采用购买的IL-11抗体(Abcam,ab130083)与IL-11蛋白之间的结合作为阳性对照。其次，通过构建稳定表达IL-11R的细胞株，并用适量的IL-11蛋白与杂交瘤上清混合后，与稳定表达有IL-11R的细胞株进行共孵育，最终通过流式检测杂交瘤上清对IL-11/IL-11R之间的阻断效果(FITC-A mean值)，其中N.C.是未加杂交瘤上清检测的值，可以视其为完全没有阻断下的值(Negative control)。表2中下划线表示选定的克隆株，经过多轮亚克隆化，最终得到单个抗体分泌细胞来源的单克隆抗体，其单克隆抗体所检测的亲和力(OD450nm)与流式阻断结果见表3。
表2：Elisa亲和实验及流式竞争实验检测结果
Figure PCTCN2022111608-appb-000017
Figure PCTCN2022111608-appb-000018
注：表2中的N/A表示：无数据
表3：Elisa亲和实验及流式竞争实验检测结果
样品编号 OD450 FITC Mean 样品编号 OD450 FITC Mean
3-6A4 1.584 3036 3-5B2-2G4 1.65 19751
5-20E11-5F2 1.596 3109 5-21G12-1D3 1.528 3362
5-20E11-1A2 1.634 3635 3-5B2-7A4 1.636 20958
3-15D5-3A6 1.688 10944 3-9E1-3A2 1.544 9067
3-7C9-1A6 1.601 3148 3-15D5-6A1 1.511 13639
3-2B5-3D2 1.614 15289 N.C.(流式) N/A 18433
注：表3中的N/A表示：无数据；因3-6A4在亚克隆化后的细胞经测序与亚克隆化前的序列一致，故其编号与表2中的编号相同。
实施例4 单克隆抗体序列测定
取1*10^6～5*10^6个杂交瘤细胞，用Takara MiniBEST Universal RNA Extraction Kit试剂盒提取总RNA，用PrimeScript RT Reagent Kit(Takara公司)试剂盒反转录出cDNA。以上步骤按照厂家提供的说明书进行。抗体重链可变区(VH)和轻链可变区(VL)基因扩增引物设计主要参考Ig-Primer Sets(Novagen公司，货号：69831-3)中的引物对，由华大基因公司合成。从cDNA第一链中扩增重链可变区和轻链可变区基因，克隆到pMD18-T载体中，转化大肠杆菌DH5α后，挑取克隆由华大基因公司测序获得重链可变区(VH)和轻链可变区(VL)基因序列。测序得到的抗体序列如下SEQ ID NO:101-122所示。
Figure PCTCN2022111608-appb-000019
Figure PCTCN2022111608-appb-000020
Figure PCTCN2022111608-appb-000021
Figure PCTCN2022111608-appb-000022
Figure PCTCN2022111608-appb-000023
上述SEQ ID NO:101和112所组成的抗体被称为3-6A4，上述SEQ ID NO:102和113所组成的抗体被称为5-20E11-5F2，上述SEQ ID NO:103和114所组成的抗体被称为3-5B2-2G4，上述SEQ ID NO:104和115所组成的抗体被称为3-5B2-7A4，上述SEQ ID NO:105和116所组成的抗体被称为3-2B5-3D2，上述SEQ ID NO:106和SEQ ID NO:117所组成的抗体被称为3-7C9-1A6，上述SEQ ID NO:107和SEQ ID NO:118所组成的抗体被称为3-9E1-3A2，上述SEQ ID NO:108和SEQ ID NO:119所组成的抗体被称为3-15D5-3A6，上述SEQ ID NO:109和SEQ ID NO:120所组成的抗体被称为3-15D5-6A1，上述SEQ ID NO:110和SEQ ID NO:121所组成的抗体被称为5-20E11-1A2，上述SEQ ID NO:111和SEQ ID NO:122所组成的抗体被称为5-21G2-1D3。
实施例5 抗体亚型鉴定
将优选抗体株对应的单克隆杂交瘤细胞株采用常规方法注射到Balb/C小鼠中进行腹水抗体的生产及纯化。利用纯化的抗体进行抗体亚型分析，抗体亚型检测方法采用成熟的试剂盒进行检测(Proteintech,KMIM-2)，结果如下表4所示。
表4：
Figure PCTCN2022111608-appb-000024
实施例6 单克隆抗体表达纯化
发明人将优选的单抗隆抗体株3-6A4及5-20E11-5F2序列采用基因工程的方法构建至常规的表达载体中，然后在CHO细胞中进行表达。表达完成后采用Protein G层析柱(EzFast Protein G 4FF，博格隆)对收集的细胞培养液进行纯化，平衡液为20mM PBS，0.15M NaCl，pH7.4，洗脱液为0.1M甘氨酸pH3.0±0.2，收集目标吸收峰下蛋白洗脱液，用PBS缓冲液超滤后，取部分样品进行SDS-PAGE电泳检测，并采用SEC-HPLC方法测定多聚体含量。抗体的多聚体含量均在3％以内。
实施例7 抗体竞争性阻断IL-11与IL-11R结合
为了检测优选抗体3-6A4及5-20E11-5F2的竞争结合情况，发明人通过在Elisa检测板中包被适量的IL-11R-His蛋白，过夜孵育。然后通过加入适量的IL-11-Fc蛋白，并与不同浓度梯度的抗体进行共孵育。最后采用HRP耦联的Goat anti IgG-Fc二抗(ThermoFisher，31413)及TMB显色底物进行检测。发明人检测到优选抗体及6D9A1(Abcam,ab130083)均能不同程度的抑制IL-11与IL-11R的结合，结果如图1所示。
实施例8 抗体的交叉反应
IL-6细胞因子家族成员包括IL-6,IL-11,CNTF,CLC,LIF,CT-1,OSM,IL-27,IL-31。IL-11属于IL-6细胞因子家族成员。发明人采用常规Elisa方法包被重组IL-6(Sinobiological-10395-HNAE),IL-27(R&D systems,2526-IL-010),LIF(Peprotech,300-05),CLC(R&D systems,962-CL-050),IL-11(LSBio,LS-G132887-10),OSM(Genscript-z03132),IL-31(Peprotech,200-31),CNTF(R&D systems,257-NT-010),CT-1(R&D systems,612-CD-010)蛋白，并检测了优选抗体3-6A4及5-20E11-5F2与IL-6细胞因子家族成员的结合情况。结果表明优选抗体除与IL-11细胞因子结合外，不与IL-6细胞因子家族其他成员发生交叉反应，Elisa结果(OD450nm)如下表5所示。
表5：抗体株3-6A4及5-20E11-5F2与IL-6细胞因子家族不同成员的结合检测
Figure PCTCN2022111608-appb-000025
实施例9 IL-11单克隆抗体与抗原结合的亲和力测定
应用生物膜干涉技术(BLI)测定，采用Ni-NTA传感器捕获抗原IL-11(His tag)，优选抗体株3-6A4及5-20E11-5F2作为分析物，分析物稀释7个浓度梯度，结合时间为60s，解离时间为1000s。缓冲液为20mM PBS，0.15M NaCl，0.1％BSA，0.05％Tween 20，pH7.4。BLI动力学/亲和力软件分析，获得优选抗体与对应hu-IL-11配体的解离常数(kdis)及亲和力常数(KD)，具体结果见下表6，其中，本实施例采用Pall ForteBio(Octet QKe)仪器测定抗体对抗原的亲和力。
表6：
Figure PCTCN2022111608-appb-000026
实施例10 抗体竞争性阻断活性检测
为了评估优选Anti-huIL-11抗体3-6A4及5-20E11-5F2与IL-11的结合，以及能否阻断配体与受体间的结合，发明人构建了稳定表达全长IL-11R的细胞株membrane-IL-11R CHO，利用流式方法检测候选抗体及商业化抗体6D9A1(Abcam,ab130083)对IL-11与IL-11R的阻断活性。首选适量的带有Fc标签的IL-11重组蛋白与不同梯度的抗体共孵育15mins，然后再与1E+05个membrane-IL-11R CHO细胞孵育。检测采用的二抗为耦联有FITC荧光基团的二抗(Goat-anti-Fc-FITC)(Abcam,ab98529)，竞争结合结果表明3-6A4抗体中和活性IC50约为7μg/mL；5-20E11-5F2抗体中和活性IC50约为21μg/mL，并且候选抗体的阻断活性明显优于6D9A1克隆株抗体的阻断活性，结果如图2所示。
实施例11 抗体株抑制肺成纤维细胞HFL-1向纤维化转化
HFL-1细胞作为肺成纤细胞，当被促纤维化因子如TGF-beta1(TGFβ)诱导后会大量表达IL-11细胞因子，最终向纤维化转化。当HFL-1细胞向纤维化转化过程中，标志物ACTA2蛋白会不断累积。在此实例中，发明人采用TGF-beta1细胞因子诱导HFL-1细胞向纤维化转化的同时加入优选3-6-A4及5-20E11-5F2抗体株时，培养48h后，收取细胞进行western检测分析，可以看到，相较于同时加入IgG(Mouse anti IgG control,Invitrogen,Catalog#02-6502)组，不同浓度的优选抗体均能够抑制HFL-1细胞向纤维化转化(ACTA2蛋白表达量降低)，如图3及对应的统计分析图4。
实施例12 抗体在C57BL/6小鼠中抑制肺纤维化
在由博来霉素诱导C57BL/6小鼠肺纤维化的动物模型中，具体来说，将10周齡的C57BL/6进行戊巴比妥麻醉剂全身麻醉后，经手术曝露出小鼠肺的主气管，然后采用滴注或雾化的方式向气管内注入适量的博来霉素。小鼠经过七天的博来霉素诱导后，肺部纤维化基本呈现。在第八天时，发明人按每隔一天的方式进行注射优选抗体5-20E11-5F2或同型对照抗体lgG。连续注射14天后，将小鼠处死取其肺，进行肺部的炎症损伤检测以及纤维化评分检测。此药效模型下，可以看到候选抗体株可以减缓小鼠肺纤维化，结果如图5和图6所示。
实施例13 IL-11抗体的人源化改造
采用互补决定区嫁接法(CDR grafting)来进行上述实施例IL-11抗体的人源化。首先寻找与鼠源抗体的轻、重链可变区同源性最高的人胚系抗体(germline antibody)序列，抗体重链可变区人源化选取的胚系为IGHV1-46*01，轻链可变区人源化选取IGKV3-11*01。保留鼠源抗体的CDR区，将鼠源抗体的框架区(framework)序列用人胚系抗体的框架区序列替换。其次，建立鼠源抗体Fv片段结构模型，在PDB抗体数据库中进行BLAST，选定结构INQB作为模板进行同源建模。逐个对比人源抗体与相应鼠源抗体结构模型中的每个不同氨基酸位点，选择不符合FR结构规范的氨基酸、位于FR内核的氨基酸、位于VH-VL界面的氨基酸，回复突变为鼠源序列。
获得的IL-11人源化抗体的重链可变区和轻链可变区的序列如SEQ ID NO：75～78所示和SEQ ID NO：79～82所示。
IL-11人源化抗体重链可变区序列(VH1):
Figure PCTCN2022111608-appb-000027
IL-11人源化抗体重链可变区序列(VH2):
Figure PCTCN2022111608-appb-000028
IL-11人源化抗体重链可变区序列(VH3):
Figure PCTCN2022111608-appb-000029
IL-11人源化抗体重链可变区序列(VH4):
Figure PCTCN2022111608-appb-000030
IL-11人源化抗体轻链可变区序列(VL1):
Figure PCTCN2022111608-appb-000031
IL-11人源化抗体轻链可变区序列(VL2):
Figure PCTCN2022111608-appb-000032
IL-11人源化抗体轻链可变区序列(VL3):
Figure PCTCN2022111608-appb-000033
IL-11人源化抗体轻链可变区序列(VL4):
Figure PCTCN2022111608-appb-000034
合成编码IL-11人源化抗体的轻链和重链的核酸序列，并插入到真核表达载体pcDNA3.4中，将4个重链序列的载体分别与4个轻链序列的载体配对，共转染至哺乳动物细胞CHO中进行表达，得到scFv形式的单链抗体。一周后收集包含有scFv抗体的上清液进行亲和力检测。
实施例14 IL-11人源化单链抗体(scFv)亲和力排序
用Biacore测定实施例13获得的各人源化单链抗体表达上清scFv与IL-11的结合，其中，所述人源化单链抗体中的连接肽为GGGGSGGGGSGGGGS(SEQ ID NO:123)，根据解离常数kd(1/s)进行排序，优选出3个解离常数kd(1/s)最小的人源化抗体。测定方法包括如下步骤：以10μL/min的流速捕获人源化单链抗体。切换流速到30μL/min，将400nM的IL-11抗原流经样品通道，结合时间为180s，解离时间为600s。最后用10mM甘氨酸缓冲液再生芯片。结果如表7所示，人源化单链抗体“VH1+VL2”-scFv、“VH1+VL3”-scFv、“VH1+VL4”-scFv与IL-11解离的速度比其他的人源化单链抗体慢。
表7：IL-11人源化抗体与IL-11抗原的亲和力
Figure PCTCN2022111608-appb-000035
Figure PCTCN2022111608-appb-000036
实施例15 优选IL-11人源化抗体的表达纯化
将优选的IL-11人源化抗体“VH1+VL2”、“VH1+VL3”、“VH1+VL4”、以及5-20E11-5F2分别进行全长序列的构建(含鼠源野生型IgG1的Fc片段)，获得包含抗体全长的pxC17.4&pxC18.4表达载体并转染至哺乳动物细胞CHO中进行表达。表达完成后采用Protein A层析柱对收集的细胞培养液进行纯化，平衡液为20mM PBS，pH7.4，洗脱液为0.1M甘氨酸pH3.0±0.2，收集目标吸收峰下蛋白洗脱液，用PBS缓冲液超滤后，取部分样品用HPLC测定浓度及进行SDS-PAGE电泳检测。
实施例16 优选IL-11人源化抗体的亲和力检测
本实验使用表面等离子共振(SPR)生物传感器Biacore T200(GE Healthcare)测定实施例15获得的所述各优选人源化全长(IgG形式)抗体样品与IL-11的结合。测定方法包括如下步骤：以10μL/min的流速捕获所述全长人源化抗体。切换流速到30μL/min，将不同浓度的IL-11抗原(400nM，200nM，100nM，50nM，25nM，12.5nM，6.25nM)流经样品通道和参比通道，结合时间为180s，解离时间为600s。最后用10mM甘氨酸缓冲液再生芯片。如表8所示，“VH1+VL3”构建的全长人源化抗体的亲和力优于另外两个全长人源化抗体，与5-20E11-5F2构建的全长嵌合抗体相比亲和力略有降低。
表8：优选人源化抗体与IL-11抗原的亲和力
抗体 Analyte ka(1/Ms) kd(1/s) KD(M) Rmax(RU) Chi 2(RU 2)
VH1+VL2 IL-11 4.98E+04 5.97E-04 1.20E-08 114.1 4.03E-01
VH1+VL3 IL-11 5.26E+04 4.74E-04 9.03E-09 105.6 3.71E-01
VH1+VL4 IL-11 5.43E+04 5.45E-04 1.00E-08 63.26 1.25E-01
5-20E11-5F2 IL-11 6.76E+04 4.33E-04 6.41E-09 72.58 6.99E-02
其中，表8中“VH1+VL2”、“VH1+VL3”和“VH1+VL4”均为人源化全长(IgG形式)抗体。
“VH1+VL2”全长人源化抗体序列的重链为SEQ ID NO:85，轻链为SEQ ID NO:90；“VH1+VL3”全长人源化抗体序列的重链为SEQ ID NO:85，轻链为SEQ ID NO:91；“VH1+VL4”全长人源化抗体序列的重链为SEQ ID NO:85，轻链为SEQ ID NO:92。
实施例17 优选人源化抗体的热稳定性实验
通过MicroCal TM VP-Capillary DSC系统对实施例15获得的所述各全长的人源化或嵌合抗体进行热稳定性分析测定。用PBS缓冲液将抗体稀释至0.4mg/mL，测定条件为：升温速率90℃/h，温度范围25-110℃。结果如表9和图7所示，人源化抗体“VH1+VL3”与5-20E11-5F2相比，Tm值提高了4.5℃。
表9：优选人源化抗体Tm值
抗体 Thalf DH TmOnset Tm1 Tm2
5-20E11-5F2 5.61 949000 63.38 72.99 84.46
VH1+VL2 4.54 981000 65.51 71.91 85.52
VH1+VL3 4.54 819000 67.6 77.49 85.23
VH1+VL4 4.54 809000 68.25 77.34 85.08
其中，表9和图7中“VH1+VL2”、“VH1+VL3”和“VH1+VL4”均为人源化全长(IgG形式)抗体；图7中“VH+VL”为5-20E11-5F2全长抗体。
实施例18 优选人源化抗体竞争性阻断IL-11与IL-11R结合
为了评估优选人源化抗体能否阻断IL-11与IL-11R的结合。发明人构建了稳定表达IL-11R的细胞株membrane-IL-11R CHO。测定方法包括如下步骤：取浓度为100μg/mL的IL-11-Fc蛋白10μL/孔加入到96孔U型板，与90μL梯度稀释的实施例15获得的所述全长嵌合抗体5-20E11-5F2和“VH1+VL2”、“VH1+VL3”和“VH1+VL4”构建的全长的人源化抗体(100μg/mL、10μg/mL、1μg/mL、0.1μg/mL、0.01μg/mL、0.001μg/mL)室温孵育1h。然后与1.5*10^5个membrane-IL-11R CHO细胞(100μL/孔)室温孵育1h后，1500rpm离心5min，用PBS清洗3次，加入1:400稀释的流式二抗Goat-anti-Fc-FITC(Abcam，ab98529)，200μL/孔，4℃孵育1h，1500rpm离心5min，用PBS清洗3次，重悬细胞于180μLPBS中，流式上机检测。结果绘制S曲线见图8，结果表明人源化抗体的阻断活性与鼠源抗体基本一致。
表10：人源化抗体竞争性阻断IL-11与IL-11R结合的EC50
抗体编号 5-20E11-5F2 VH1+VL2 VH1+VL3 VH1+VL4
EC50(μg/mL) 7.537 11.57 10.46 12.48
其中，表10和图8中“VH1+VL2”、“VH1+VL3”和“VH1+VL4”均为人源化全长(IgG形式)抗体。
实施例19 优选人源化抗体抑制肝成纤维细胞LX-2向纤维化转化
LX-2细胞是人肝星状细胞，当被促纤维化因子如TGF-beta1(TGF-β)诱导后会大量表达IL-11细胞 因子，最终向纤维化转化。当LX-2细胞向纤维化转化的过程中，标志物ACTA2蛋白会不断累积。在此实例中，发明人以3*10^5个细胞/孔的密度将LX-2细胞铺于6孔板培养10h至细胞贴壁。用无血清DMEM培养基饥饿培养12h后，加入5ng/mL的TGF-beta1细胞因子进行刺激，同时分别加入梯度稀释的实施例15获得的“VH1+VL3”构建的全长的人源化抗体(“VH1+VL3”mAb 5μg/mL或10μg/mL)。培养24h后，吸出培养基，每孔加入200μL RIPA裂解液(碧云天，P0013B)，进行进行western blot检测分析。结果如图13所示，不同浓度的人源化抗体均能抑制LX-2细胞向纤维化转化(Collagen IA蛋白表达量降低)，表明优选人源化抗体能很好抑制LX-2细胞向纤维化转化。
实施例20 优选抗体在SD大鼠中抑制肺纤维化
本实施例将化药(HEC585(盐酸伊啡尼酮)、BIBF1120)以及实施例15获得的5-20E11-5F2构建的全长嵌合抗体(CPD-1)、“VH1+VL3”构建的全长人源化抗体(CPD-2，即所述“VH1+VL3”mAb)进行动物实验，其中，以IgG抗体作为对照。具体实验操作如下：
采用2.5％异氟烷将大鼠进行麻醉，手术打开主气管后，注射适量的溶媒或博来霉素进行造模，其中，采用博来霉素(Bleomycin)诱导以构建SD大鼠单侧肺纤维化的动物模型，手术完成后观察大鼠恢复情况。手术后的第8天进行灌胃或腹腔给药进行治疗14天，具体动物分组及给药剂量汇总如下表11所示。当给予人源化抗体治疗后，可以明显观察到人源化抗体减缓大鼠肺的炎症反应及纤维化进程。
表11：5-20E11-5F2与人源化抗体(VH1+VL3)大鼠药效实验方案
Figure PCTCN2022111608-appb-000037
其中，表11中“VH1+VL3”为人源化全长(IgG形式)抗体。
治疗结束后，取大鼠的肺进行石蜡包埋后切片，并进行HE染色及Masson三染色。其中HE染色主要指示肺组织中炎症的浸润水平。Masson三染色主要用于评价肺组织纤维化的水平。结果图9与图10均表明优选人源化抗体能很好的降低博来霉素所引起的大鼠肺炎症损伤及纤维化。此外在高剂量组下，人源化抗体对大鼠肺炎症损伤效果明显优于5-20E11-5F2抗体。
实施例21 优选人源化抗体的亲和力成熟
利用噬菌体展示技术进行抗体亲和力成熟。将优选人源化抗体VH1+VH3构建成VH-Linker-VL形式的ScFv，以此为模板进行噬菌体建库并进行多轮淘选，经过ELISA及流式检测挑选出亲和力优于原始序列的候选物。构建CDR突变库和随机突变库，两种库分别构建，在淘选时将噬菌体混合。(1)设计引物构建4个CDR突变子库：分别为(VH-CDR3、VL-CDR3、VH-CDR1)；(VH-CDR3、VL-CDR3、VH-CDR2)；(VH-CDR3、VL-CDR3、VL-CDR1)；(VH-CDR3、VL-CDR3、VL-CDR2)，四个子库在酶切前进行等比例混合。同时构建随机突变库，通过易错酶经过多轮PCR得到含有随机突变位点的ScFv序列，将这些序列构建到噬菌粒载体上即为随机突变库。验证库的质量及抗体多样性分析。(2)抗体淘选：对CDR突变库和随机突变库使用不同浓度的IL-11抗原，进行多轮液相筛选能结合IL-11的噬菌体，并通过噬菌体ELISA检测和流式检测其亲和特性，然后经测序获得亲和活性较好的克隆共有14个。流式检测以及测序结果见表12。(3)优选出3个克隆(1E12、2E2、1F12)，利用编码该3个克隆以及野生型IgG1Fc片段的核酸构建全长抗体PXC17.4&PXC18.4表达载体，转染CHO细胞，经protein A纯化得到抗体。
表12：亲和力成熟测序结果
Figure PCTCN2022111608-appb-000038
实施例22 亲和力成熟后抗体的亲和力检测
用Fortebio测定实施例21获得的各亲和力成熟的1E12、2E2、1F12全长抗体样品(编号分别MIL-11mab-01、MIL-11mab-02、MIL-11mab-03)与IL-11的结合。测定方法包括如下步骤：用AHC传感器捕获人源化抗体。分析物为不同浓度的IL-11抗原(125nM，62.5nM，31.3nM，15.6nM，7.81nM，3.91nM，)，结合时间为200s，解离时间为1000s。最后用10mM甘氨酸(pH1.7)缓冲液再生传感器。如表13所示，亲和力成熟后的抗体MIL-11mab-02、MIL-11mab-03亲和力比人源化抗体提高了10倍，与“VH1+VH3”和野生型IgG1Fc片段构建的全长人源化抗体亲和力相当。
表13：亲和力成熟抗体亲和力结果
Ligand Analyte ka(1/Ms) kd(1/s) KD(M)
VH1+VH3 IL-11 2.14E+05 6.09E-04 2.84E-09
MIL-11mab-01 IL-11 5.01E+05 5.84E-04 1.17E-09
MIL-11mab-02 IL-11 5.35E+05 1.30E-04 2.42E-10
MIL-11mab-03 IL-11 5.80E+05 1.52E-04 2.62E-10
其中，表13中“VH1+VL3”为人源化全长(IgG形式)抗体。
实施例23 亲和力成熟抗体流式细胞术体外结合活性鉴定
发明人构建了稳定表达IL-11的细胞株membrane-IL-11CHO。取梯度稀释的实施例21获得的亲和力成熟抗体(100μg/mL、10μg/mL、1μg/mL、0.1μg/mL、0.01μg/mL、0.001μg/mL)与1.5*10^5个membrane-IL-11CHO细胞室温孵育1h后，用PBS清洗3次，加入流式二抗Goat-anti-Fc-FITC(Abcam，ab98529)，流式上机检测。结果绘制S曲线见图11，图中Hu-IL11mab是指“VH1+VH3”构建的人源化抗体，结果表明亲和力成熟抗体的结合活性与人源化抗体基本一致。
实施例24 亲和力成熟抗体流式细胞术体外阻断活性鉴定
为了评估优选人源化抗体以及亲和力成熟抗体阻断IL-11与IL-11R的结合之间的差异，发明人使用稳定表达IL-11R的细胞株membrane-IL-11R CHO。测定方法包括如下步骤：取浓度为10μg/mL的IL-11-His蛋白10μL/孔加入到96孔U型板，与90μL梯度稀释的优选人源化抗体(100μg/mL、10μg/mL、1μg/mL、0.1μg/mL、0.01μg/mL、0.001μg/mL)室温孵育1h。然后与1.5*10^5个membrane-IL-11R CHO细胞(100μL/孔)室温孵育1h后，1500rpm离心5min，用PBS清洗3次，加入1:200稀释的流式二抗PE-anti-His Tag(Biogend，362603)，200μL/孔，4℃孵育1h，1500rpm离心5min，用PBS清洗3次，重悬细胞于180μLPBS中，流式上机检测。结果绘制S曲线见图12，图中Ch-IL11mab表示“VH1+VH3”构建的嵌合型抗体，结果表明亲和力成熟抗体的阻断活性与人源化抗体基本一致。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等 的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (13)

  1. 一种能够特异性识别IL-11的抗体或其抗原结合片段，其特征在于，所述抗体包括：
    分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；和
    分别如SEQ ID NO:37、38和39或者与SEQ ID NO:37、38和39具有至少95％同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列；和
    重链框架区包括FL1、FL2、FL3和FL4，所述重链框架区FL1、FL2、FL3和FL4分别具有SEQ ID NO:67、68、69和70所示的氨基酸序列，
    QVQLVQSGAEVKKPGASVKVSCKAS(SEQ ID NO:67)，
    LGWVRQAPGX1GLEWX2GH(SEQ ID NO:68)，
    NYNEKFKGRX3TX4TX5DTSTSTVYMELSSLRSEDTAVYX6C(SEQ ID NO:69)，
    WGQGTLVTVSS(SEQ ID NO:70)，
    其中，X1为Q或H，X2为M或I，X3为V或A，X4为M或L，X5为R或A，X6为Y或F；和
    轻链框架区包括FL1、FL2、FL3和FL4,所述轻链框架区FL1、FL2、FL3和FL4分别具有SEQ ID NO:71、72、73和74所示的氨基酸序列，
    EIVLTQSPATLSLSPGERATLSCRAS(SEQ ID NO:71)，
    MNWX7QQKPGQAPRLLIY(SEQ ID NO:72)，
    NQGSGIPARFSGSGSGTDFTLTISSLEPEDFAX8YX9C(SEQ ID NO:73)，
    FGGGTKLEIK(SEQ ID NO:74)，
    其中，X7为Y或F，X8为V或M，X9为Y或F。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体具有如SEQ ID NO：75～78任一项所示氨基酸序列的重链可变区。
  3. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体具有如SEQ ID NO：79～82任一项所示氨基酸序列的轻链可变区。
  4. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体含有重链恒定区和轻链恒定区的至少之一，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  5. 根据权利要求4所述的抗体或其抗原结合片段，其特征在于，所述抗体恒定区的全长序列如SEQ ID NO:83或84所示。
  6. 根据权利要求5所述的抗体或其抗原结合片段，其特征在于，所述抗体具有SEQ ID NO:85～88任一项所示氨基酸序列的重链和具有SEQ ID NO:89～92任一项所示氨基酸序列的轻链。
  7. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～6任一项所述的抗体或其抗原结合片段。
  8. 一种表达载体，其特征在于，携带权利要求7所述的核酸分子。
  9. 一种重组细胞，其特征在于，所述重组细胞携带权利要求7所述的核酸分子，或者表达权利要求1～6任一项所述的抗体或其抗原结合片段。
  10. 一种药物组合物，其特征在于，含有权利要求1～6任一项所述的抗体，权利要求7所述的核酸分子，权利要求8所述的表达载体或权利要求9所述的重组细胞。
  11. 权利要求1～6任一项所述的抗体、权利要求7所述的核酸分子、权利要求8所述的表达载体或权利要9求所述的重组细胞、权利要求10所述的药物组合物在制备药物中的用途，所述药物用于治疗或者预防肺纤维化，非酒精性脂肪肝炎，慢性心力衰竭或由成纤维细胞向纤维化转化所导致的疾病。
  12. 一种检测IL-11的试剂盒，其特征在于，包括权利要求利要求1～6所述的抗体。
  13. 权利要求1～6任一项所述的抗体、权利要求7所述的核酸分子、权利要求8所述的表达载体或权利要求9所述的重组细胞在制备试剂盒中的用途，所述试剂盒用于检测IL-11或者诊断IL-11相关的疾病。
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