WO2012116595A1 - 抗肿瘤坏死因子α的人源化抗体 - Google Patents

抗肿瘤坏死因子α的人源化抗体 Download PDF

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WO2012116595A1
WO2012116595A1 PCT/CN2012/071079 CN2012071079W WO2012116595A1 WO 2012116595 A1 WO2012116595 A1 WO 2012116595A1 CN 2012071079 W CN2012071079 W CN 2012071079W WO 2012116595 A1 WO2012116595 A1 WO 2012116595A1
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amino acid
antibody
seq
replaced
necrosis factor
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PCT/CN2012/071079
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English (en)
French (fr)
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朱保国
陶德胜
孙乃超
金宜慧
刘瑞贤
周若芸
李强
彭育才
张经纬
胡振湘
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珠海市丽珠单抗生物技术有限公司
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=46757361&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2012116595(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to BR112013021860-6A priority Critical patent/BR112013021860A2/pt
Priority to AU2012222738A priority patent/AU2012222738B2/en
Priority to KR1020137025433A priority patent/KR101576559B1/ko
Priority to EP12752672.1A priority patent/EP2682405A4/en
Priority to JP2013516997A priority patent/JP6054864B2/ja
Application filed by 珠海市丽珠单抗生物技术有限公司 filed Critical 珠海市丽珠单抗生物技术有限公司
Priority to RU2013139480/10A priority patent/RU2549700C1/ru
Priority to CA2827799A priority patent/CA2827799C/en
Priority to NZ615477A priority patent/NZ615477B2/en
Priority to US13/405,144 priority patent/US8658171B2/en
Priority to ARP120100640A priority patent/AR085501A1/es
Publication of WO2012116595A1 publication Critical patent/WO2012116595A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C07K2317/70—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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a humanized antibody against at least one anti-tumor necrosis factor-a (TNFa) or a fragment thereof, including a specific portion or variant thereof, and a nucleic acid encoding the anti-TNFa humanized antibody, a complementary nucleic acid thereof, and a vector Host cells, methods for their preparation, and compositions, kits and uses thereof comprising the anti-TNFa humanized antibodies.
  • TNFa anti-tumor necrosis factor-a
  • TNFa Human tumor necrosis factor-a
  • monocytes and macrophages a pro-inflammatory cytokine produced by monocytes and macrophages. It originally produced a 26kDa precursor protein with the N-terminus in the cell and the C-terminus in the extracellular domain.
  • TNFa transmembrane TNFa
  • Pennica et al first cloned the TNF-a gene cDNA in 1984 and concluded that the human TNF- ⁇ molecule consists of 157 amino acids with a molecular weight of approximately 17KD (Pennica D, et al, Nature 1984; 312 :724).
  • Human TNFa has two molecular forms, namely TNFa and TNFP.
  • TNFa is produced by activated macrophages or monocytes, which can cause hemorrhagic necrosis of tumor tissue, also known as cachexia; TNF- ⁇ is mainly secreted by activated T lymphocytes. , both have similar heat.
  • TNFa acts on receptors on the surface of tumor cells, and by cell recognition, binding, endocytosis into lysosomes, lysosomes and proteases are highly activated, leading to cell death, which plays a role in immune response, inflammation and damage Important role, mainly affects the regulation of cell proliferation and apoptosis, not only has cytotoxicity, cell lysis, induction of apoptosis, inhibition of proliferation, but also promotes differentiation of myeloid leukemia cells into macrophages. The phagocytic capacity of granulocytes.
  • TNFa activates the immune system to enhance the body's immunity, and plays a key role in the defense system against host microbial invasion and tumor suppression.
  • TNFa can produce a variety of pathological damages together with other inflammatory factors, thereby inhibiting or neutralizing the activity of TNFa at different levels, so that it can not reach the receptor, thereby avoiding the consequences of signal transduction.
  • Such a chimeric antibody is a non-human antibody variable region that is grafted into a human antibody constant region, retaining the amino acid sequence of the heavy chain and light chain variable regions of the non-human antibody (see, PCT Publication No. WO 92/16553 to Daddona, RE, et al., U.S. Patent No. 5,919, 452 to Le, J. et al., PCT Publication No. WO2005/047329 to Kang, Heui H, et al.
  • Jin Yihui et al. Chinese Patent Publication No. CN101177453
  • the degree of murineization in antibodies remains high, which may lead to varying degrees of HAMA response, manifested as skin mucosal reactions, allergies, arrhythmias and angina, renal insufficiency, and may even cause coma in severe cases.
  • the clinical application of antibodies is greatly limited.
  • the chimeric antibody acts as a heterologous protein in the human body, and can cause the immune rejection of the heterologous protein by the immune system of the body, that is, a human anti mouse antibody (HAMA) response reaction.
  • HAMA human anti mouse antibody
  • the murine monoclonal antibody is often rapidly cleared in the human body, and its half-life is also short. Repeated medication may even lead to severe anaphylactic shock in the patient.
  • these foreign antibodies may be attacked by immune antibodies, with the result that they are neutralized before they appear to be effective.
  • the humanized antibody can be prepared by genetic engineering technology, that is, the murine antibody heavy chain variable region,
  • the Complementarity determining reign (CDRs) of the light chain variable region are separately transplanted into the human antibody framework region (FR), and the humanized antibody thus obtained is as close as possible to the human sequence in its structure. At the same time, it maintains its similar CDR conformation to the parent non-human antibody.
  • the engineered humanized antibody is derived from a decrease in the non-human amino acid sequence of the parent, which on the one hand maintains the ability of the antibody to recognize the antigen; on the other hand, greatly reduces the murine nature.
  • the immunogenicity of the antibody increases the safety of the antibody in clinical applications.
  • the present invention provides a humanized antibody which is safer and more reliable than the mouse chimeric antibodies of the prior art and which has a longer half-life and more significant function in humans. Summary of the invention
  • An aspect of the invention provides at least one anti-tumor necrosis factor humanized monoclonal antibody and specific complementarity determining region (CDR), heavy or light chain variable region, heavy or light chain constant region as described herein Zone, frame zone or any part thereof.
  • Antibodies of the invention may include or It is derived from any mammal, such as, but not limited to, human, mouse, rat, rodent, primate, or any combination thereof, and the like.
  • An antibody of the invention specifically binds to a particular epitope of at least one TNF protein, a subunit thereof, a fragment, a portion, or any combination thereof.
  • the at least one epitope may comprise at least one antibody binding region.
  • the at least one antibody may optionally comprise at least one complementarity determining region (CDR) (such as a heavy chain variable region or a light chain variable region) and/or at least one constant or variable framework region (FR) or Any part of it.
  • CDR complementarity determining region
  • FR constant or variable framework region
  • the amino acid sequence of the at least one antibody may further optionally comprise an insertion, deletion or conservative substitution of at least one amino acid residue.
  • a further aspect of the invention provides at least one nucleic acid molecule comprising a polynucleotide encoding at least one of the anti-tumor necrosis factor humanized antibodies herein, or with said at least one anti-tumor encoding herein
  • the polynucleotides of the necrosis factor humanized antibody are complementary or hybridized, and the antibody comprises at least one specific sequence, domain, portion or variant thereof.
  • a further aspect of the invention provides a recombinant vector comprising the anti-tumor necrosis factor humanized antibody nucleic acid molecule, a host cell comprising the nucleic acid and/or a recombinant vector, and the nucleic acid, vector and/or host cell Preparation methods and/or applications.
  • At least one antibody of the invention has at least one activity such as, but not limited to, neutralizing the toxicity, inhibition and/or competition of TNF with a receptor and/or other monoclonal antibodies such as, but not limited to, Ada.
  • a further aspect of the invention provides the use of an antibody and/or composition of the invention for inhibiting human hTNFa activity, wherein the hTNFa-related disease may be, for example, sepsis, autoimmune disease, malignancy, pulmonary dysfunction, transplantation Rejection, bacterial meningitis, cerebral malaria, AIDS and AIDS-related syndrome (ARC), cytomegalovirus infection secondary to transplantation.
  • hTNFa-related disease may be, for example, sepsis, autoimmune disease, malignancy, pulmonary dysfunction, transplantation Rejection, bacterial meningitis, cerebral malaria, AIDS and AIDS-related syndrome (ARC), cytomegalovirus infection secondary to transplantation.
  • a further aspect of the invention provides the use of an antibody and/or composition of the invention in the manufacture of a medicament for the diagnostic analysis of hTNFa, wherein the anti-tumor necrosis factor humanized monoclonal antibody is also detectable molecule Marking and/or non-labeling, including radioisotopes; fluorescent labels; various enzyme substrate labels.
  • the analytical methods include competitive binding assays, direct or indirect sandwich assays, or immunoprecipitation assays.
  • a further aspect of the invention provides at least one composition
  • a composition comprising the present invention An anti-tumor necrosis factor humanized antibody and/or nucleic acid encoding the same, one or more adjuvants selected from the group consisting of, but not limited to, pharmaceutically acceptable carriers, excipients, diluents, additives, and the like.
  • the composition may optionally further comprise at least one other antibody, nucleic acid, adjuvant, or any combination thereof.
  • a further aspect of the invention provides a kit comprising a predetermined amount of reagents and instructions, the reagents comprising an antibody, nucleic acid and/or composition of the invention.
  • the kit also includes the substrate and cofactor required for the enzyme.
  • the kit may further include, but is not limited to, stabilizers, buffers, and the like.
  • FIG 1 shows the effect of antibody neutralization of TNFa on U937 cells.
  • Figure 2 shows the degree of swelling of joints induced by type II collagen in rats.
  • Figure 3A shows the in vivo score of arthritis in Tgl97 mice
  • Figure 3B shows the pair in the treatment group.
  • FIG. 3C shows the effect of the treatment group on arthritis score (AS) and histopathology (HS) scores in Tgl97 mice. detailed description
  • An antibody of the invention comprises an antibody amino acid sequence of the invention encoded by any suitable polynucleotide, or any isolated or prepared antibody.
  • the humanized antibody or antigen-binding fragment preferably binds to human tumor necrosis factor and thereby partially, substantially or wholly neutralizes at least one biological activity of human tumor necrosis factor, thereby inhibiting binding of TNF to TNF receptor Mediated signaling processes and physiological processes.
  • the humanized antibody of the present invention may be of any type (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may comprise a kappa or lambda light chain and an alpha, ⁇ , gamma, epsilon or delta heavy chain.
  • At least one antibody of the invention binds to at least one specific epitope of at least one TNF protein, a subunit thereof, a fragment, a portion, or any combination thereof.
  • the at least one anti-tumor necrosis factor humanized monoclonal antibody of the present invention has the following amino acid sequence, wherein the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1 And the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 2: SEQ ID NO: 1 :
  • complementarity determining region CDR-H1 of the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 3;
  • CDR-H2 is the amino acid sequence set forth in SEQ ID NO: 4;
  • CDR-H3 is SEQ ID NO: The amino acid sequence shown in 5; wherein A of amino acid 16 in the framework region FR-H1 can be replaced by E, S of amino acid 17 can be replaced by T, and I of amino acid 20 can be replaced by V (SEQ ID NO: 11);
  • FR- The K of amino acid 3 in H2 can be replaced by R, the G of amino acid 9 can be replaced by S (SEQ ID NO: 12); the T of amino acid 3 in FR-H3 can be replaced by V, and the E of amino acid 7 can be replaced by D, amino acid
  • the V of 10 can be replaced by T
  • the F of amino acid 14 can be replaced by Y
  • the S of amino acid 19 can be replaced by T
  • T of amino acid 27 can be replaced by V (SEQ ID NO
  • L of amino acid 11 in the framework region FR-L1 can be replaced by M
  • the R of amino acid 18 can be replaced by E
  • the M of amino acid 21 can be replaced by I (SEQ ID NO: 1 4)
  • W of amino acid 13 in FR-L2 can be replaced by L (SEQ ID NO: 15)
  • S of amino acid 4 in FR-L3 can be replaced by A
  • L of amino acid 22 can be replaced by V
  • a of amino acid 27 Can be replaced by F (SEQ ID NO: 16).
  • the heavy chain constant region sequence of the humanized antibody of the invention is the heavy chain constant region sequence of human IgG1.
  • the light chain constant region sequence of a humanized antibody of the invention is the light chain constant region sequence of a human antibody.
  • the amino acid sequence of the anti-tumor necrosis factor humanized monoclonal antibody may replace one or more amino acid residues, preferably 1-3 amino acids, by insertion, deletion or conservation. The residue is modified.
  • Modified or mutated monoclonal antibodies differ in amino acid sequence. Preferred variants are modified from the monoclonal antibodies of the invention described above by conservative substitution of the amino acids. The conservative substitution refers to the replacement of a specified amino acid with another amino acid of similar nature. The following amino acids exemplified in a non-limiting manner are considered to be conservatively replaceable
  • a monoclonal antibody functionally equivalent according to the invention is a variant in which one or more amino acid residues, preferably 1-3 amino acid residues, are conservatively substituted.
  • Conservative substitution refers to the substitution between the aromatic amino acids Ala, Val, Leu and lie; the substitution between the hydroxyl residue Ser and Thr; the substitution between the acidic residues As and Glu; the amide residue Mutual substitution between Asn and Gin; mutual substitution between basic residues Lys and Arg; mutual substitution between aromatic residues Phe and Tyr.
  • the present invention discloses amino acid sequences having at least 50% homology to the amino acid sequences disclosed herein, or fragments thereof and functionally equivalent amino acid sequences.
  • the amino acid sequence has at least 75% homology, more preferably at least 85% homology, more preferably at least 90% homology to the amino acid sequences SEQ ID NO: 1 and 2 provided herein. More preferably, it is at least 95% homologous, more preferably at least 97% homologous, and most preferably at least 99% homologous.
  • the anti-hTNFa antibody may be a full-length antibody (e.g., having an intact human Fc region), or an antibody fragment (e.g., Fv, scFv, Fab, Fab, and (Fab,) 2) and the like.
  • the antibody can be labeled with a detectable label, immobilized on a solid support, and/or coupled to a heterologous compound (e.g., a cytotoxic substance).
  • Fab is obtained by treating IgG antibody molecules with protease/papain.
  • protease/papain This is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, wherein in the fragment obtained by papain (the amino acid residue is cleaved at position 224 of the H chain), about half of the N-terminal side of the H chain and the whole The L chains are bonded together by disulfide bonds.
  • the Fab of the present invention can also be produced by inserting the DNA of the Fab encoding the antibody into a prokaryotic expression vector or a eukaryotic expression vector to express Fab.
  • Fab' is an antibody fragment having antibody binding activity, which is produced by incision of a disulfide bond of the (Fab') 2 hinge region, and has a molecular weight of about 50,000.
  • the Fab of the present invention can also be obtained by encoding an antibody.
  • the DNA of the Fab' fragment is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the vector is introduced into a prokaryote or eukaryote to express a Fab.
  • (Fab') 2 is an antibody fragment having antigen-binding activity and having a molecular weight of about 100,000.
  • Fab fragments obtained by treating an IgG antibody with a protease/pepsin (cutting the H chain at the 234th amino acid residue)
  • Fab The antibody fragments joined together by disulfide bonds in the hinge region are slightly larger.
  • the (Fab,) 2 of the present invention can be produced by treating an antibody with pepsin. Further, (Fab,) 2 of the present invention can also be produced by linking a Fab with a thioether bond or a disulfide bond.
  • scFv is an antibody fragment having antigen-binding activity, using an appropriate peptide linker connecting together a chain and a V H chain V L.
  • the production of the scFv of the present invention can be carried out by obtaining a VH and V] cDNA encoding the antibody, constructing a DNA encoding the scFv, inserting the DNA of the scFv encoding the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and then expressing the expression vector Introduction of scFv into prokaryotes or eukaryotes.
  • Nucleic acid sequences of the invention encoding at least 70-100% of contiguous amino acids of at least one of SEQ ID NOS: 1-16, a particular fragment, variant or consensus sequence thereof, may be as described herein or Methods of the invention are known in the art to obtain nucleic acid molecules of the invention encoding at least one anti-TNF antibody.
  • the nucleic acid molecule of the present invention may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or may be in the form of DNA, including, but not limited to, cDNA and genomic DNA produced by cloning or synthesis, or any combination thereof.
  • DNA can be double-stranded or single-stranded. Any portion of at least one strand of DNA or RNA may be a coding strand, also referred to as a sense strand, or may be a non-coding strand, also referred to as an antisense strand.
  • a nucleic acid molecule of the invention comprising a nucleic acid encoding an anti-TNF antibody can include, but is not limited to, a nucleic acid encoding an amino acid sequence of an antibody fragment; a coding sequence of an intact antibody or a portion thereof; an antibody, fragment or portion a coding sequence, as well as other sequences, such as at least one signal leader peptide or a coding sequence of a fusion peptide, with or without other coding sequences as previously mentioned, such as at least one intron, with other non-coding Sequences, including, but not limited to, non-coding 5' and 3' sequences, such as transcription, mRNA processing, including splicing and polyadenylation signals (eg, ribosome binding and stability of mRNA), not a sequence of translations; encoding other amino acids, such as providing other functions Other sequences of capable amino acids.
  • a sequence encoding an antibody can be fused to a marker sequence, for example, to a coding sequence that facilitates purification of a fusion antibody comprising an antibody fragment or portion.
  • Nucleic acids of the invention also include polynucleotides that selectively hybridize to the polynucleotides described herein.
  • the nucleic acids of the present invention can be prepared by (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or a combination thereof, which are well known in the art.
  • Nucleic acids of the invention such as DNA, RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any cloning method known to those skilled in the art.
  • an oligonucleotide probe that selectively hybridizes to a polynucleotide of the invention under stringent conditions is used to identify a desired sequence in a cDNA or genomic DNA library. Isolation of RNA and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art.
  • a cDNA or genomic library can be screened using probes based on the polynucleotide sequences of the present invention.
  • Probes can be hybridized to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • hybridization of varying degrees of stringency can be used in the assay, and any of the hybridization or wash media can be stringent. When the hybridization conditions are more stringent, the complementarity between the probe and the target sequence must be higher in order to form a duplex.
  • the degree of stringency can be controlled by one or more conditions in the presence of temperature, ionic strength, pH, and partial denaturing solvents such as guanamine.
  • the polarity of hybridization can be varied by, for example, operating the concentration of guanamine in the range of 0% - 50%.
  • the degree of complementarity (sequence equivalence) required for detectable binding will vary depending on the stringency of the hybridization medium and/or wash medium.
  • the degree of complementarity is preferably 100% or 70-100%, or any range or any of them.
  • small sequence variations in probes and primers can be compensated for by reducing the stringency of hybridization and/or wash media.
  • RNA or DNA Methods for amplifying RNA or DNA are well known in the art.
  • Known methods for amplifying RNA or DNA include, but are not limited to, polymerase chain reaction (PCR) and related amplification methods and RNA-mediated amplification.
  • PCR polymerase chain reaction
  • the nucleic acids of the invention can also be prepared by direct chemical synthesis according to known methods. Chemical synthesis generally produces single-stranded nucleotides that can be converted to double strands by hybridization with complementary sequences or by polymerization with a DNA polymerase using a single strand as a template. DNA. Construction of humanized monoclonal antibody expression vector
  • a plasmid (pHu- VH ) containing the humanized antibody heavy chain variable region sequence VH gene fragment was used as a template, and a 5' primer FVHX (5, -CGCGCAAG-CTTCCTCGAG-3, SEQ ID NO: 17) and 3 were used.
  • ' Primer RVCG (5 ' CGATGGGCCCTTGGTGGA3 ' SEQ ID NO: 18) gave a 5' fragment containing the heavy chain variable region (V H ) of the humanized antibody and 7 of the 5' end of the human 0 1 heavy chain constant region (C) Amino acid gene.
  • RNA prepared from human leukocytes was selected with the appropriate 5' primer HuCGF (5 '-ACCAAGGGCCCATCGGTCTTC-3 '; SEQ ID NO: 19) and 3' primer HUCGE (5 '-CGGAATTCTCATTTACCC GGAGACAGGGA 3, SEQ ID NO : 20)
  • HuCGF 5'-ACCAAGGGCCCATCGGTCTTC-3 '; SEQ ID NO: 19
  • HUCGE 5'-CGGAATTCTCATTTACCC GGAGACAGGGA 3, SEQ ID NO : 20
  • a gene containing a human IgGi heavy chain constant region (C) coding sequence was obtained by reverse transcription reaction and PCR.
  • a fragment containing the heavy chain variable region ( VH ) of the humanized antibody and the above human gene were subjected to PCR using a 5' primer (FVHX, SEQ ID NO: 17) and a 3' primer (HUCGE, SEQ ID NO: 20), the heavy chain variable region (v H ) of the humanized antibody and the human c gene fragment are ligated to obtain a gene fragment having a heavy chain coding sequence and having a length of about 1400 b.
  • This gene fragment is treated with endonuclease Hind III and EcoRl and inserted into a vector such as PUC19 (ref: Yanisch-Perron, C, Vieira, J. and Messing, J. (1985) Gene, 33, 103-119.).
  • RNA prepared from human leukocytes was selected with the appropriate 5' primer HuCKF (5'-GTG GCT GCA CCA TCT GTC TTC-3, SEQ ID NO: 22) and 3' primer HUCKB (5'-TGC GGA TCC).
  • HuCKF 5'-GTG GCT GCA CCA TCT GTC TTC-3, SEQ ID NO: 22
  • HUCKB 5'-TGC GGA TCC
  • a gene containing the human kappa light chain constant region (CK) coding sequence was obtained by reverse transcription and PCR.
  • VL light chain variable region
  • FVHX 5' primer
  • UCKB 3' primer
  • VL humanized light chain variable region
  • VL humanized light chain variable region
  • HCKB 3' primer
  • This gene fragment is endonuclease Hind III and Bam After HI treatment, an vector such as PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33, 103-119.) is inserted.
  • the expression vector plasmid contains the cytomegalovirus early gene promoter-enhancer required for high level expression in mammalian cells. At the same time, the vector plasmid contains a selectable marker gene to confer ampicillin resistance in bacteria and G418 resistance in mammalian cells.
  • the vector plasmid contains the DHFR gene, and in a suitable host cell, the chimeric antibody gene and the DHFR gene can be co-amplified with Methotrexate (MTX, Sigma) (see, for example, Axel, R., et al. U.S. Patent No. 5,179,017; Kaufman, R. and Sharp, R, J. Mol. Biol. 159:601-621, 1982).
  • MTX Methotrexate
  • the invention also relates to host cells engineered with a recombinant vector, and at least one anti-TNF antibody produced by recombinant techniques well known in the art.
  • the polynucleotide may optionally be linked to a vector containing a selectable marker for propagation in a host.
  • the plasmid vector is introduced into a precipitate, such as calcium phosphate precipitates, or a complex with charged lipids.
  • Suitable media and culture conditions for the host cells are well known in the art. Suitable vectors are readily understood by those skilled in the art. Vector constructs can be introduced into a host cell by calcium phosphate transfection, DEAE-dextran-mediated transfection, anionic lipid-mediated transfection, electroporation, transduction, infection, or other known methods.
  • the host cell of the anti-tumor necrosis factor humanized monoclonal antibody of the present invention is derived from Chinese Hamster Ovary, transfected with a plasmid containing an anti-TNFa gene internal code, and then a series of rigorous, specific The screening process is obtained, including drug screening, gene amplification and single cell cloning to establish the final cell line.
  • the host cell Chinese hamster ovary cell line CHO HUAT 132 of the present invention was deposited with the China Center for Type Culture Collection on March 7, 2011, under the accession number CCTCC NO.: C201117.
  • the cells of this cell line can be suspended in serum-free medium; when cultured in a 2-liter fermenter, the level of anti-TNFa secreted in the culture medium after the end of the 16-20 day culture period will Not less than one gram per liter (1 g/L).
  • the anti-TNFa produced by this cell line is a humanized monoclonal antibody. Binding activity to TNFa
  • the anti-tumor necrosis factor humanized monoclonal antibody of the present invention has only a specific affinity with recombinant human tumor necrosis factor (rhTNFa, a target molecule) and does not cross-link with any other protein molecule.
  • rhTNFa recombinant human tumor necrosis factor
  • a similar affinity to adalimumab (adalimumab) can be measured in a competitive test for the affinity of its target molecule.
  • the anti-tumor necrosis factor humanized monoclonal antibody of the present invention also has activity to neutralize the toxicity exhibited by rhTNFa to L929-targeted cells, and its EC50 value is similar to the EC50 value of adalimumab, ranging from 20.4 ng/mL to 50 ng. Within the /mL concentration range.
  • the anti-hTNFa antibody can be used for the treatment and prevention of TNFa-related diseases, wherein the hTNFa-related diseases can be, for example, sepsis, autoimmune diseases, malignant tumors, pulmonary dysfunction, transplant rejection, bacterial meningitis, cerebral malaria, AIDS AIDS-related syndrome (ARC), transplantation of cytomegalovirus infections, and further discussion of the use of the antibodies and antibody portions of the invention in the treatment of hTNFa-related diseases
  • Tumor necrosis factor has been established in the pathology of sepsis, and its biological effects include hypotension, myocardial depression, vascular leakage syndrome, organ necrosis, etc. (see, for example, U.S. Patent No. 5,231,024), thus humanizing the present invention.
  • the antibody and antibody portions can be used to treat sepsis in any clinical setting, including septic shock, endotoxic shock, Gram-negative sepsis, and toxic shock syndrome.
  • TNFa has been found to be involved in activating tissue inflammation and causing joint destruction in rheumatoid arthritis (see, e.g., U.S. Patent No. 5,231,024; Moeller, A. et al. (1990) Cytokine 2: 162-169 ;), also found that TNFa is involved in the promotion of islet cell death and mediates cytotoxicity and induction of inflammatory plaques on oligodendrocytes in diabetes.
  • the humanized antibodies and antibody portions of the invention can be used to treat autoimmune diseases, Don't be autoimmune diseases related to inflammation, including rheumatoid arthritis, rheumatoid arthritis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune eyes Pigmentitis and nephrotic syndrome.
  • Tumor necrosis factor has been found to be involved in the induction of cachexia, stimulation of tumor growth, enhanced metastatic potential, and mediating cytotoxicity in malignant tumors.
  • the antibodies and antibodies of the invention can be used to partially treat malignant tumors, inhibit tumor growth or metastasis, and/or reduce cachexia secondary to malignant tumors.
  • the antibody or antibody portion can be administered systemically or topically to the tumor site.
  • Tumor necrosis factor is known to be involved in the pathophysiology of adult respiratory distress syndrome (ARDS), including stimulation of leukocyte-endothelial cell activation, cytotoxicity-directed lung cells, and induction of vascular leakage syndrome.
  • ARDS adult respiratory distress syndrome
  • pulmonary dysfunction can be treated with the antibodies and antibody portions of the invention, including adult respiratory distress syndrome, chronic pneumonia, pulmonary sarcoidosis, pulmonary fibrosis, and silicosis.
  • Intestinal dysfunction such as idiopathic inflammatory bowel disease
  • idiopathic inflammatory bowel disease can be treated using the human antibodies and antibody portions of the invention, including two syndromes, Crohn's disease and ulcerative colitis.
  • Tumor necrosis factor has been found to be a key mediator of allograft rejection and transplanted plant-versus-host disease (GVHD) and is involved in mediating inhibition of renal transplant rejection with the rat antibody OKT3 targeting the T cell receptor CD3 complex. Side reactions (see, for example, Suthanthiran, M. and Strom, TB (1994) New Engl, J. Med. 331: 365-375)
  • GVHD plant-versus-host disease
  • the antibodies and antibodies of the present invention can be used to partially treat infectious diseases, including bacterial meningitis, cerebral malaria, AIDS and AIDS-related syndromes (ARC), and transplanted secondary cytomegalovirus infections, and can also be used to alleviate infectious diseases.
  • infectious diseases including bacterial meningitis, cerebral malaria, AIDS and AIDS-related syndromes (ARC), and transplanted secondary cytomegalovirus infections.
  • Related symptoms include fever and myalgia caused by infection (eg flu) and cachexia secondary to infection (eg AIDS or ARC secondary:).
  • Analytical and diagnostic use include fever and myalgia caused by infection (eg flu) and cachexia secondary to infection (eg AIDS or ARC secondary:).
  • the antibodies of the invention can be used in any of the known analytical methods, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays. Zola, Monoclone Antibodies; A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
  • Sandwich assays involve the use of two antibodies, each of which binds to a different immunogenic site or epitope of the protein to be tested.
  • the analyte of the sample to be tested binds to the first antibody immobilized on the solid support, and then the second antibody binds to the analyte, thereby forming an insoluble tripart complex.
  • the second antibody itself may be labeled with a detectable moiety (direct sandwich assay) or by an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay).
  • one of the sandwich assays is an ELISA in which the detectable moiety is an enzyme.
  • Anti-hTNFa antibodies can also be used in diagnostic assays for hTNFa, e.g., to detect expression in specific cells, tissues or serum. This diagnostic method can be used to diagnose the cause of autoimmune diseases and the like.
  • Antibodies are usually labeled with a detectable molecule. There are a number of tags that can be used, which can be roughly classified as follows:
  • Radioisotopes such as lllln, 99Tc, 14C, 1311, 1251, 3H, 32P or 35S.
  • the radioisotope can be used, for example, by the method described in Current Protocols in Immunology, Vol. 1 and 2, Coligen et al., Wiley-Interscience, New York, New York, Pubs. (1991).
  • the antibody is labeled and the radioactivity can be determined by scintillation counting to capture the diseased site using immunoscingraphy.
  • Fluorescent labels such as rare earth chelating agents ( ⁇ chelating agents) or luciferins and their derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin and Texas Red (Texas red) ). Fluorescent labeling can utilize methods and antibodies as described, for example, in the Modern Immunology Methods above. Coupling. Fluorescence can be quantified using a fluorometer.
  • Enzyme labels include, for example, luciferase (eg, firefly luciferase and bacterial luciferase; US Patent 4,737,456), luciferin, 2,3-dihydrophthalazinediones.
  • luciferase eg, firefly luciferase and bacterial luciferase; US Patent 4,737,456
  • luciferin 2,3-dihydrophthalazinediones.
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • b-galactosidase glucoamylase
  • lysozyme sugar oxidase (eg glucose oxidation)
  • Enzymes galactose oxidase and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases
  • the enzyme-substrate composition includes, for example:
  • HRP horseradish peroxidase
  • a perhydroperoxidase as a substrate, wherein the hydroperoxidase causes a dye precursor (for example, o-phenylenediamine (OPD) or hydrochloric acid 3, 3', 5,5'-tetradecylbenzidine (TMB)) oxidation;
  • a dye precursor for example, o-phenylenediamine (OPD) or hydrochloric acid 3, 3', 5,5'-tetradecylbenzidine (TMB)
  • bD-galactosylase bD-Gal
  • chromogenic substrate eg p-nitrophenyl-bD-galactosidase
  • fluorogenic substrate 4-mercapto umbelliferone-bD-galactoside Enzyme
  • the label is indirectly coupled to the antibody.
  • the antibody can be coupled to biotin, and any of the above three types of labels can be coupled to avidin, or vice versa.
  • Biotin selectively binds to avidin, and the label can be coupled to the antibody in an indirect manner.
  • the antibody in order to indirectly couple the label to the antibody, can be combined with a small half-antibody
  • the original (e.g., digoxin) is coupled, and one of the different types of labels described above is coupled to an anti-hapten antibody (e.g., an anti-digoxigenin antibody). This results in an indirect coupling of the label to the antibody.
  • an anti-hapten antibody e.g., an anti-digoxigenin antibody
  • the anti-hTNFa antibody need not be labeled and its presence can be detected using a labeled antibody that binds to the hTNFa antibody.
  • Affinity purification reagent Affinity purification reagent
  • the antibody of the present invention can be used as an affinity purification reagent.
  • the antibody is immobilized on a solid phase such as Sephadex resin or filter paper by a method known in the art.
  • the immobilized antibody is contacted with the hTNFa-containing sample to be purified, and then the carrier is washed with a suitable solvent capable of substantially removing all other substances in the sample except for hTNFa bound to the immobilized antibody.
  • the antibody and antibody portion of the invention may be added to a pharmaceutical composition suitable for administration to a subject, wherein the pharmaceutical composition comprises an antibody of the invention and a pharmaceutically acceptable excipient, the pharmaceutically acceptable excipient including any physiology Suitable solvents, dispersion media, antibacterial agents, antifungal agents, isotonic agents, coatings, absorption delaying agents, and the like.
  • the pharmaceutical compositions of this invention may be in a variety of forms including, for example, liquid semi-solid and solid dosage forms.
  • the anti-hTNFa antibody of the present invention in a pharmaceutically acceptable dosage form can be administered to a human by a known method.
  • the method includes intravenous (eg, intravenous bolus or continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intraarterial, intrasynovial, intrathecal For injection, oral, topical or inhalation.
  • the antibody can also be suitably administered through the intratumor, around the tumor, in the wound site, or around the wound site to exert a local and systemic therapeutic effect.
  • the intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian cancer.
  • the appropriate dose of antibody will depend on the type of disease to be treated as defined above, the severity and duration of the disease, whether the antibody is administered for prophylaxis or treatment, previous treatment, patient history and The responsiveness to antibodies and the independent judgment of the attending physician.
  • the antibodies are suitable for use in a single or series of patients.
  • 1 microgram / kg to 50 mg / kg (e.g. 0.1 to 20 mg / kg) of antibody is The initial candidate dose to be used by the patient. Typical daily or weekly doses may range from about 1 mg/kg to 20 mg/kg or more, depending on the factors mentioned above. For repeated dosing over a period of several days or longer (depending on the condition), the treatment needs to continue until the desired inhibition of the disease symptoms occurs. However, other dosing regimens can also be used. The progress of the treatment can be conveniently monitored using conventional techniques and analytical methods. product
  • a product containing a material for treating the above diseases includes containers and labels.
  • Suitable containers include, for example, regular bottles, vials, syringes, and test tubes.
  • the container can be made of various materials such as glass or plastic.
  • the container contains an effective composition for treating the disease and has a sterile access port (e.g., the container can be a stoppered IV bag or vial that can be penetrated by a hypodermic needle).
  • the active ingredient in the composition is an anti-hTNFa antibody.
  • the label on or associated with the container indicates the particular condition being treated by the composition.
  • the product may also contain another container containing a pharmaceutically acceptable buffer such as a phosphate buffer, a Ringer solution, and a dextrose solution. It may also include other materials, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use, depending on the needs of the business or the needs of the user.
  • a pharmaceutically acceptable buffer such as a phosphate buffer, a Ringer solution, and a dextrose solution.
  • It may also include other materials, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use, depending on the needs of the business or the needs of the user.
  • the anti-tumor necrosis factor humanized monoclonal antibody of the present invention can be used for the preparation of a sustained release preparation.
  • Suitable sustained release formulations include, for example, semipermeable matrix bodies of solid hydrophobic polymers comprising the antibodies, which are tangible objects such as films or microcapsules.
  • Suitable sustained release matrix bodies include, for example, polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactide (U.S. Patent 3,773,919), L-glutamine.
  • Copolymer of acid and L-glutamic acid ethyl ester non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid such as Lupron DepotTM (injectable microsphere composed of lactic acid-glycolic acid copolymer and leuprolide acetate) Copolymer and poly-D-(-)-3-hydroxybutyric acid.
  • Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, and some hydrogels can release proteins in a shorter period of time. When gelatinized antibodies remain in the body for a prolonged period of time, they may denature or aggregate due to contact with water at 37 ° C, resulting in reduced biological activity.
  • a reasonable stabilization strategy can be designed. For example, if the aggregation mechanism is found to form an intermolecular SS bond through a sulfur-disulfide bond exchange reaction, stabilization can be accomplished by modifying the thiol residue, lyophilizing the acidic solution, controlling the water content, using suitable additives, and design. This is achieved with a special polymeric matrix composition. Kit
  • the antibody of the present invention may be provided in the form of a kit in which a predetermined amount of the reagent is combined with a package for carrying out the diagnostic analysis.
  • the kit will contain the substrate and cofactor required for the enzyme (e.g., a substrate precursor that provides a measurable chromophore and fluorophore).
  • other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like.
  • the reagents may be in the form of a dry powder, usually in the form of a lyophilized powder, which, including excipients, will form a reagent solution of the appropriate concentration upon dissolution.
  • Blood can be taken from the mice after 12-25 and 25-40 days by post-frame puncture without anticoagulant conditions. The blood was then allowed to coagulate at RT for 1 hour, serum was collected, and the titer was determined using TNFa ELA according to a known method. Fusion is performed when repeated injections do not result in an increase in titer. at this time, The mice were given a last booster injection with 1-400 ⁇ g TNFa diluted in 100 ⁇ saline. After 3 days, the mice were sacrificed by cervical dislocation, and the spleen was removed under aseptic conditions and immersed in 10 mL containing 1000 U/mL penicillin, 100 g/mL streptomycin, and 0.25 g/mL amphotericin B (PSA).
  • PSA amphotericin B
  • Splenocytes were harvested by aseptically perfusing the spleen with PSA-PBS, counted by trypan blue stain exclusion, and resuspended in RPMI 1640 medium containing 25 mM Hepes.
  • mice spleen cells with high levels of anti-hTNFa antibody serum identified by serum test were fused with mouse myeloma cells in a ratio of 1:1 to 10:1.
  • spleen cells are precipitated together with myeloma cells, and then resuspended in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight 1450, sigma) at 37 °C. More than two seconds. Then, 10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37 ° C) was slowly added for 1 minute or more to terminate the fusion.
  • the fused cells were centrifuged at 500-1500 rpm for 5 minutes.
  • HAT medium RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 g/mL Qingda , 2.5% Origen culture supplement (Fisher), 10% 653 adjusted RPMI 1640/Hepes medium, 50 ⁇ 2-mercaptoethanol, 100 ⁇ hypoxanthine and 16 ⁇ thymidine), and 200 ⁇ 7 well flat Spread over 15 96-well flat bottom tissue culture plates. The plates were then placed in a humidified 37 ° C incubator containing 5% CO 2 and 95% air for 7-10 days.
  • Example 2 qualification of anti-hTNFa murine antibody
  • the anti-hTNFa human antibody adalimumab was labeled with horseradish peroxidase (HRP) as a reagent.
  • HRP horseradish peroxidase
  • RhTNF (1, 0.05 g / mL) was coated on the plate, and allowed to stand overnight at room temperature. The coating solution was discarded, and the wells were blocked with 1% skim milk dissolved in phosphate buffered saline (PBS) for 0.5 hour. The wells were washed with PBS containing 0.05% Tween 20. Then a mixture of 50 ⁇ growth medium and 50 ⁇ HRP-labeled adalimumab was added to each well.
  • PBS phosphate buffered saline
  • Unlabeled adalimumab and no antibody The medium was used as a positive and negative control, respectively.
  • a murine monoclonal antibody containing a high ability to inhibit the binding of HRP-labeled adalimumab to rhTNfa was screened.
  • the wells containing the inhibition of HRP-labeled adalimumab binding to rhTNFa were amplified.
  • subcloning continue to further analyze several murine monoclonal antibodies showing this inhibition, and finally screen out the two hybridoma cells.
  • the two hybridoma cells were expanded and cultured, and purified by clear liquid to obtain mouse monoclonal antibody TM2-11. -12 with TM2-6-3.
  • TM2-11-12 Purified binding assay of purified murine monoclonal antibody TM2-11-12 with TM2-6-3, mouse antibody TM2-6-3 at concentrations up to 1 g/mL, only Can compete for about 50% of adalimumab and hTNFa
  • Another murine antibody, TM2-11-12 showed the same excellent competing ability as unlabeled adalimumab, at about 0.05 g/mL (equivalent to 3x10 ° M), it was able to compete 50% adalimumab binds to hTNf ⁇ .
  • IC 5 can be used. Values to compare the ability of antibodies to inhibit hTNFa cytotoxicity.
  • Several anti-hTNFa murine antibodies TM2-11-12, TM2- 10-20, TM2-2-2) IC 5 . Values with adalimumab IC 5 . Values ranged from 0.01 to 0.04 g/mL, i.e., all had similar ability to neutralize L929 cytotoxicity induced by rhTNFa.
  • Anti-hTNFa murine antibody TM2- 11-12 was selected for further preparation of chimeric antibodies.
  • Example 3 Cloning of the heavy and light chains of the TM2-11-12 murine antibody
  • primers (5, - butyl ( ⁇ into 00000 ⁇ into 0 00 into the butyl into 0 into 0 into 0 into -3,, SEQ ID NO:
  • SEQ ID NO: 29 is used as 5
  • the light chain variable region DNA fragment was isolated from the cDNA. These obtained DNA fragments were cloned into the TOPO TA vector and sequenced, and two types of clones were found. Approximately 3/4 of the clones showed that part of the nucleotide sequence could not be translated into a readable amino acid sequence (the sequence is not shown). Such clones are distorted light chain messenger RNAs that do not encode a functional antibody light chain protein. In addition, about 1/4 of the cloned nucleotide sequences are fully translated into a readable amino acid sequence, and such clones are derived from functional light chain messenger RNA.
  • the humanized design is to change the amino acid residues in the framework region according to the sequence of the human antibody, and design the heavy chain variable region and the light chain variable region of the humanized antibody with various modifications, through computer simulation technology, The oligonucleotide sites of the antibody heavy and light chain variable region sequences are subjected to directed mutagenesis. To increase antibody binding affinity or to reduce antibody immunogenicity.
  • a of amino acid 16 in the framework region FR-H1 can be replaced by E
  • S of amino acid 17 can be replaced by T
  • I of amino acid 20 can Replaced by V
  • K of amino acid 3 in FR-H2 can be replaced by R
  • G of amino acid 9 can be replaced by S
  • T of amino acid 3 in FR-H3 can be replaced by V
  • E of amino acid 7 can be replaced by D
  • amino acid 10 The V can be replaced by T, the F of amino acid 14 can be replaced by Y, the S of amino acid 19 can be replaced by T, and the T of amino acid 27 can be replaced by V.
  • the L of amino acid 11 in the framework region FR-L1 can be replaced by M
  • the R of amino acid 18 can be replaced by E
  • the M of amino acid 21 can be replaced by I substitution
  • W of amino acid 13 in FR-L2 can be replaced by L
  • S of amino acid 4 in FR-L3 can be replaced by A
  • L of amino acid 22 can be replaced by V
  • a of amino acid 27 can be replaced by F.
  • the amino acid sequence of the variable region of the heavy chain of the humanized antibody is designed by introducing at least one of the above amino acid modifications, and the amino acid sequence of the humanized antibody heavy chain variable region VH and the light chain variable region VL is shown in the table. 1 shows:
  • the gene fragment of the humanized antibody light chain variable region (F001VL) is prepared by artificial synthesis, and the preparation procedure can be obtained by reversely translating the amino S sequence of the light chain variable region with its relative genetic code.
  • the nucleotide sequence is added to the Kozak sequence and the light chain leader sequence at 5, and the gene fragment of the light chain variable region is prepared by artificial synthesis.
  • This gene fragment was cloned into an appropriate vector to obtain a pHu-VL1 plasmid.
  • the pHu-VL1 plasmid was used as a template, and the 5' fragment was obtained using the 5' primer FVHX (SEQ ID NO: 17) and the 3' primer VKCKO (SEQ ID NO: 21), and the light chain containing the humanized antibody was variable. region (V L) and the human ⁇ light chain constant region (CK) 5 'end of the gene fragments of 7 amino acids.
  • the human K light chain constant region was obtained by reverse transcription and PCR using RNA prepared from human leukocytes with the appropriate 5' primer HuCKF (SEQ ID NO: 22) and 3' primer HUCKB (SEQ ID NO: 23).
  • CK A gene encoding a sequence.
  • V u a fragment containing the light chain variable region (V u ) of the humanized antibody and the above human CK gene were subjected to PCR using a 5' primer (FVHX, SEQ ID NO: 17) and a 3' primer (HUCKB, SEQ ID NO: 23), the humanized light chain variable region (V U ) and the human CK gene fragment are ligated to obtain a gene fragment containing a light chain coding sequence of about 700 bp in length.
  • This gene fragment is treated with endonuclease Hind III and Bam HI and inserted into a vector such as PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33, 103-119.), A light chain protein used to express a humanized antibody. The sequence of this gene fragment was verified by DNA sequencing.
  • a gene fragment of the humanized antibody heavy chain variable region (E001VH) is prepared by artificial synthesis, and the preparation procedure can be obtained by reversely translating the amino S sequence of the heavy chain variable region with its relative genetic code.
  • the nucleotide sequence is added to the Kozak sequence and the heavy chain leader sequence at 5, and the gene fragment of the heavy chain variable region is prepared by artificial synthesis.
  • This gene fragment was cloned into an appropriate vector to obtain a pHu-VH1 plasmid.
  • the 5' primer FVHX (SEQ ID NO: 17) and the 3' primer RVCG (SEQ ID NO: 18) were used to obtain a 5' fragment, and the heavy chain containing the humanized antibody was variable. gene region (V H1) and the human IgGi heavy chain constant region (C yl) 5 'end of the 7 amino acids.
  • RNA containing human leukocytes was used to obtain a human IgGi heavy chain constant by reverse transcription reaction and PCR using the appropriate 5' primer HuCGF (SEQ ID NO: 19) and 3' primer HUCGE (SEQ ID NO: 20). The gene encoding the sequence.
  • a fragment containing the heavy chain variable region ( VH ) of the humanized antibody and the above human Cy gene were subjected to PCR using a 5' primer (FVHX, SEQ ID NO: 17) and a 3' primer (HUCGE, SEQ ID NO: 20), the heavy chain variable region ( VH1 ) of the humanized antibody and the human gene fragment are ligated to obtain a gene fragment having a heavy chain coding sequence and having a length of about 1400 bp.
  • This gene fragment is treated with endonuclease Hind III and EcoRl and inserted into a vector such as PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33, 103-119.)
  • PUC19 a vector such as PUC19 (ref: Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33, 103-119.)
  • the cDNA encoding the heavy and light chains obtained in the above method was inserted into a vector of pcDNA3 (purchased from Invitrogen USA, Carlsbad, CA USA) to construct a pHu_anti-HlL1-TNFa humanized expression vector.
  • the expression vector plasmid contains the cytomegalovirus early gene promoter-enhancer required for high level expression in mammalian cells.
  • the vector plasmid contains a selectable marker gene to confer ampicillin resistance in bacteria and G418 resistance in mammalian cells.
  • the vector plasmid contains the DHFR gene, and in a suitable host cell, the chimeric antibody gene and the DHFR gene can be co-amplified with Methotrexate (MTX, Sigma) (see, for example, Axel, R., et al. U.S. Patent No. 5,179,017; Kaufman, R. and Sharp, P., J. Mol. Biol. 159:601-621, 1982).
  • MTX Methotrexate
  • the above constructed recombinant expression vector plasmid is transfected into a mammalian host cell line to express an anti-hTNFa humanized antibody.
  • a preferred host cell line is dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells (see, e.g., U.S. Patent No. 4,818,679 to Chasin, L. et al.).
  • DHFR dihydrofolate reductase
  • CHO Chinese hamster ovary
  • Example 7 Killing effect of antibody neutralizing TNFa on L929 cells
  • L929 cells were trypsinized, centrifuged, resuspended in 10% FCS 1640 medium, counted, added to the 1 to 11 column of 96-well plate at a certain concentration, and then added with appropriate concentration of TNFa in the 1 to 10 column of the 96-well plate. , prepared with a first well concentration of 0.4 ug/mL, a 2-fold gradient of adalimumab (purchased from Abbott Laboratories), an amino acid unmodified chimeric antibody AT (CE)-1, and prepared according to the methods of Examples 5 and 6.
  • U937 cells in good condition were counted, and the cell concentration was adjusted to 3.75 ⁇ 10 4 cells/well in 10% FCS 1640 medium, and added to a 96-well culture plate at 75 ⁇ M/well.
  • the AT132 standard and the test sample were diluted with a cell culture medium containing 120 ng/mL TNFa, and the concentration of the first well was 600 ng/mL, and the gradient was 1.5 times. After the translation, the sample was 25 ⁇ M/well. Add to a 96-well plate and incubate for 40 hours in a 37 ° C carbon dioxide incubator.
  • FIG 1 shows the effect of antibody neutralization of TNFa on U937 cells.
  • Fig. 1 The results of Fig. 1 show that TNFa is basically killing cells at the very low concentration of AT132. With the increase of AT132 concentration, the action of TNFa is gradually antagonized. When the concentration of AT 132 is about 80 ng/mL, the killing of TNFa The effect was essentially completely antagonized, so there was a significant dose-dependent, according to the results of multiple trials, the median effective concentration of 30 ng/mL TNFa was 24.1 ng/mL on average.
  • Example 9 AT132 Affinity Determination
  • Biacore X 100 assay, Biacore X 100 kinetic/affinity software analysis, indirect capture method, goat anti-human IgG Fc polyclonal antibody was coupled to the surface of CM5 chip as capture molecule by Amine Coupling Kit, and AT132 was calculated by calculation.
  • control adalimumab diluted to a certain concentration as a ligand, TNFa as an analyte Analyze the 5 concentrations of the analyte, each concentration as a cycle, first run 3 cycles with HBS-EP buffer, design an analyte concentration of 0 to run 2 cycles, and finally design a repeated analyte concentration run 1 cycle. The entire process was run for 11 cycles, and a curve was drawn for each cycle.
  • the kinetic/affinity data of the tested antibody adalimumab was analyzed by Biacore X100 Kinetic/Affinity Analysis software.
  • the AT132 dissociation constant (Kd) is 1.19x lO_ u M, that is, the affinity constant (Ka) is The adalimumab dissociation constant is 1.08x l0 - 1Q M, ie the affinity constant is 9.3 ⁇ 10 9 ⁇ .
  • Example 10 AT132 binds to murine TNFa and monkey TNFa
  • a solution of hydrogen hydroxide was prepared by mixing 40.0 g of sodium hydroxide with 1000.8 g of water for injection.
  • the buffer is then prepared by dissolving the following pre-weighed ingredients (as described above) in approximately 90% water for injection: mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate , sodium dihydrogen phosphate, sodium chloride and sorbitan polyoxyethylene (20) ether oleate.
  • the pH of the solution was adjusted with 1 M sodium hydroxide prepared as described above. After the addition of sodium hydroxide, the final weight of water is added.
  • the buffer was then filtered through a filter membrane (hydrophilic polyvinylidene fluoride, 0.22 ⁇ pore size) into a sterile container.
  • the filter medium used is filter sterilized ammonia.
  • the filtered buffer solution was added to the thawed and combined antibody concentrate (active ingredient of the pharmaceutical preparation) prepared as follows.
  • the antibody (concentrate) was thawed in a water bath prior to preparation of the pharmaceutical formulation.
  • a 34.207 g antibody concentrate was used, which corresponds to 2.0 kg of protein at a concentration of 60 mg protein/mL protein concentrate.
  • the concentrate had a density of 1.0262 g/mL. Any protein concentration in the range of 25.655-37.316 can be used , which corresponds to a protein concentration of 50-80 mg/mL in the protein concentrate.
  • the buffer is added under agitation until the final weight of the total solution is reached.
  • Example 12 Acute toxicity test of AT132 mice
  • Test sample AT132 freeze-dried powder, specifications: 20 mg / bottle; adjuvant reference: AT132 buffer (containing histidine, trehalose); Solvent: sterile water for injection.
  • Groups of test animals and doses 60-6 Kunming (KM) mice, 4-22 weeks old, 18-22 g, half male and half female, SPF grade. The above animals were randomly divided into 3 groups, 20 in each group, half male and half female. Single administration, observed for 14 days. Dosage and route of administration as shown in Table 3:
  • AT132 powder was administered to mice with single intravenous and subcutaneous injection at a dose of 500 mg/kg. No significant toxicity was observed. The maximum tolerated dose (MTD) was greater than 500 mg/kg.
  • Example 13 AT132 induces lethal protection of rhTNFa in D-galactosamine-sensitized mice 51 C57BL/6 mice, weighing 20.0 ⁇ 2.0 g, were divided into 6 groups (see Table 4). Each of the mice in Groups 2-5 was intraperitoneally injected with 0.25 mL of ATI 32 solution, wherein the dose of each group of mice in the second group was 5.2 g per unit of AT132, and the dose of each group of mice in Group 3 was administered.
  • mice in group 4 In the case of ATI 32, it is 26 g/head, the dose of mice in group 4 is 52 ⁇ M per unit, and the dose of mice in group 5 is 26 g per unit in AT132; Group 1 mice were injected intraperitoneally with 0.25 mL of citrate buffer pH 5.63. Group 6 mice were injected intraperitoneally with 0.25 mL of human IgGi HuIgGi (negative control), and HulgGi was 26 g. /only.
  • mice were intraperitoneally injected with 0.25 mL of rhTNFa (Primegene, batch number 1030109021) and D-aminogalactose mixture solution, and group 5 mice were intraperitoneally injected with 0.25 mL buffer. The mice were observed for 48 hours of lethality, and the calculated survival rate is shown in Table 4.
  • RESULTS The survival rate of the buffer and HulgGi mice was 0, while AT132 dose-dependently protected rhTNFa and D-galactosamine-sensitized mice. Therefore, ATI 32 has a protective effect on rhTNFa-induced lethality in D-galactosamine-sensitized mice.
  • Example 14 Effect of AT132 on type II collagen-induced arthritis in rats Wistar female rats were randomly divided into 4 groups, 10 in each group. They were blank control group, proinflammatory control group (ie model group), ATI 32 l mg/kg group and AT 132 5 mg/kg group.
  • the experimental data were expressed as mean and standard deviation (s), and analyzed by t test. The results are shown in Figure 2.
  • the AT132 1 mg/kg group showed obvious inhibitory effect, and it had a good effect at the beginning of the 19th day, and the best effect was 63.10%. After that, the inhibitory effect gradually decreased, and there was still a good effect on the 28th day.
  • the inhibition rate was 55.71%; AT 132 5 mg/kg group showed obvious inhibitory effect, and it had significant effect from the 19th day.
  • the redness and swelling of the rat's foot joint gradually subsided, the activity became normal, and the effect continued until the 28th day.
  • the inhibition rate was the highest on the 21st day, which was 47.27%, and the others were about 40%.
  • Example 15 Pharmacodynamic study of AT132 on T g 197 mouse arthritis model Tgl97 transgenic mice (purchased from Cyagen Biosciences) were used in this experiment. The mice were divided into 6 groups of 10 mice each, male and female. half. Specifically: Group 1: AT 132 1 mg/kg; Group 2: Solvent group (buffer containing citric acid and sodium chloride); Group 3: AT132 30 mg/kg; Group 4: AT132 10 mg/kg) Group 5: Adalimumab 10 mg/kg; Group 6: ATI 32 3 mg/kg. Another 4 Tgl97 mice were selected as a blank control group.
  • mice Three-week-old Tgl97 mice were intraperitoneally injected with AT132 twice a week to 10 weeks of age; AT132 was diluted to the desired concentration with buffer before administration, and the dose of each test group administered was ⁇ ⁇ / g body weight, observing the degree of arthritis in mice, pathologically scoring the joints of the mice, and calculating the inhibition rate according to the arthritis score and the pathological score.
  • 0.5 onset of arthritis (joint paws are slightly swollen, the appearance is normal, the mice can support the body's weight, the overall flexibility / escape ability is normal, the grip is the greatest);
  • 1.0 mild arthritis (the joints are swollen and deformed, the paws are red and swollen, the appearance is normal, the mice can support the body's weight, the overall flexibility / escape ability is normal, the grip is the most);
  • mice numbered Conl-Con4 mice
  • the experimental mice were sacrificed at 10 weeks of age, and the ankle joint sections were taken.
  • the microscopic histopathological scores of arthritis were evaluated blindly, with a rating of 0-4, as follows:
  • Pathology score inhibition rate % E o - E" X 100%
  • Parasitic slices of human nasal polyps and four sets of normal human tissues (donors A, B, C, D, by The China National Institute for Food and Drug Control, provided by the National Center for Drug Safety Evaluation and Monitoring, is divided into three groups: the experimental group (biotin marker AT132), the positive control group (biotin-labeled adalimumab), and the negative control group. Buffer PBS), observe the coloration of tissue sections after tissue cross-reaction.
  • the experimental results showed that the negative control group of human nasal polyps and normal human tissues were not stained.
  • the biotin-adalimumab positive control group showed weak to moderate staining with macrophages of human nasal polyps, macrophages of normal human lymph nodes, and alveolar macrophages of the lung; the remaining tissues were not stained.
  • Biotin-AT132 experimental group and macrophages of human nasal polyps showed weak to moderate staining; normal human tissue cross-reactivity had similar results with adalimumab.
  • Paraffin sections of human nasal polyps and three sets of normal cynomolgus monkey tissues were divided into three groups, namely the experimental group.
  • donors A, B, C provided by China National Institute for Food and Drug Control, National Center for Drug Safety Evaluation and Monitoring
  • Biotin label AT132 positive control group
  • biotin-labeled adalimumab negative control group
  • coloration of tissue sections after cross-reaction of tissues was observed.
  • the experimental results showed that there was no staining in the negative control group and the normal cynomolgus monkey tissues.
  • the biotin-adalimumab positive control group and the human nasal polyp macrophages showed weak to moderate staining; normal cynomolgus monkey tissues showed no staining.
  • Weak to moderate staining was observed in the biotin-AT132 experimental group and human nasal polyp macrophages.
  • the cross-reaction of normal cynomolgus monkey tissue has similar results with adalimumab.

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Description

抗肿瘤坏死因子 α的人源化抗体 技术领域
本发明涉及至少一种抗肿瘤坏死因子 -a (TNFa)或其片段的人源化 抗体， 包括其特定部分或变体， 以及编码所述抗 TNFa人源化抗体的核 酸、 其互补核酸、 载体、 宿主细胞及其制备方法和包含所述抗 TNFa人 源化抗体的组合物、 试剂盒及其应用。 背景技术
人肿瘤坏死因子 -a(TNFa)是由单核细胞和巨噬细胞产生的一种促 炎性细胞因子， 最初生成的是 26kDa的前体蛋白， N端在胞内， C端在 胞外， 称为跨膜型 TNFa , Pennica等 1984年首先克隆了 TNF-a基因 cDNA,并推导出人 TNF-α分子由 157个氨基酸组成，其分子量约为 17KD (Pennica D, et al, Nature 1984; 312:724)。 人 TNFa有两种分子形式， 即 TNFa和 TNFP. TNFa是由激活的巨噬细胞或单核细胞产生，可引起肿 瘤组织出血坏死， 也称恶病质素； TNF-β主要由活化的 T淋巴细胞分泌， 两者有相似致热性。 TNFa作用于瘤细胞表面的受体， 通过对细胞的识 别、 结合、 内吞进入溶酶体， 致溶酶体和蛋白酶类高度活化导致细胞 死亡， 它在免疫反应、 炎症和对损伤反应中起重要作用， 主要影响细 胞增殖和细胞凋亡的调节， 不仅对肿瘤细胞有细胞毒性、 细胞溶解、 诱导细胞凋亡、抑制增殖等作用，还能够促进髓样白血病细胞向巨噬 细胞分化， 提高中性粒细胞的吞噬能力。
适量的 TNFa能激活免疫系统从而增强机体的免疫力， 能够在宿主 抵抗微生物入侵和抑制肿瘤产生的防御系统中起着关键作用。 但过量 的 TNFa表达时， 可以与其它炎性因子一起产生多种病理损伤， 因此可 在不同水平上抑制或中和 TNFa的活性， 使其不能达到受体， 进而避免 信号传导引发的后果。
为试图克服使用非人源抗体造成的相关问题， 通过构建的人 -鼠嵌 合抗体来降低 HAMA引发的机体免疫原性， 已成为较为有效的治疗策 略。 此种嵌合抗体就是将非人源抗体可变区移植到人抗体恒定区， 保 留了非人源抗体原有的重链、 轻链可变区的氨基酸序列（参见， Daddona, RE等的 PCT公开 W092/16553号， Le, J.等人的美国专利 5 , 919, 452号， Kang, Heui H等 PCT公开 WO2005/047329号， 金伯泉等的 中国专利公开 CN1544466A号）， 金宜慧等中国专利公开 CN101177453 号提供了一种新的与人肿瘤坏死因子结合和嵌合抗体， 此种嵌合抗体 相对于非人源抗体， 其免疫原性虽有所降低， 但是， 由于嵌合抗体中 鼠源化程度依然较高， 从而可能导致不同程度的 HAMA应答反应， 具 体表现为皮肤黏膜反应、 变态反应、 心律失常和心绞痛、 肾功能不全， 严重时甚至可能造成昏迷， 此类嵌合抗体的临床应用受到了很大的限 制。
通过临床试验证明， 此种嵌合抗体作为异种蛋白作用于人体后， 可引起机体免疫系统对该异种蛋白的免疫排斥反应， 即人抗鼠抗体 (Human anti mouse antibody, HAMA)应答反应， 该反应会使鼠源单抗 在人体内往往被快速清除， 其半衰期也较短， 重复用药甚至可能导致 病人严重的过敏性休克。 而且， 这些外来抗体可能受到免疫抗体的攻 击， 其结果使得他们在呈现药效之前被中和掉。
发明人在上述专利技术基础上进行了开发， 为了尽可能的降低在 嵌合抗体中的鼠源部分， 可利用基因工程技术制备人源化抗体， 即， 将鼠源抗体重链可变区、 轻链可变区的互补决定区 (； Complementarity determining reign , CDRs)分另 ll移植 人抗体骨架区 (Framework region , FR), 由此获得的人源化抗体在其结构上尽可能接近人类序列的同时， 也能维持其与亲本非人源抗体相似的 CDR构象。相对于亲本非人源抗 体和嵌合抗体， 改造后的人源化抗体来源于亲本非人源氨基酸序列减 少， 一方面保持了抗体识别抗原的能力； 另一方面， 极大降低了鼠源 性抗体的免疫原性， 从而提高了抗体在临床应用的安全性。
因此， 本发明提供了一种比现有技术中的小鼠嵌合抗体更加安全 可靠的， 在人体内半衰期更长、 功能更显著的人源化抗体。 发明内容
本发明的一方面提供了如本文所描述的至少一种抗肿瘤坏死因子 人源化单克隆抗体及特定的互补决定区 (CDR)、 重链或轻链可变区、 重 链或轻链恒定区、 构架区或其任意部分。 本发明的抗体可以包括或来 源于任何哺乳动物， 例如， 但不限于人、 小鼠、 大鼠、 啮齿类动物、 灵长类动物或其任意组合等。
本发明的抗体特异性地结合至少一种 TNF蛋白、 其亚基、 片段、 部分或其任意组合的特定表位。 所述至少一种表位可以包含至少一种 抗体结合区。 所述至少一种抗体可以任选地包含至少一种互补决定区 (CDR) (如重链可变区或轻链可变区）和/或至少一种恒定或可变构架区 (FR)或其任意部分。 所述至少一种抗体的氨基酸序列可以进一步任选 包含至少一种氨基酸残基的插入、 缺失或保守性置换。
本发明再一方面提供了至少一种核酸分子， 所述核酸分子包含编 码至少一种本文的抗肿瘤坏死因子人源化抗体的多核苷酸， 或者与所 述编码至少一种本文的的抗肿瘤坏死因子人源化抗体的多核苷酸相互 补或者相杂交， 所述抗体包含至少一种其特定序列、 结构域、 部分或 变体。
本发明的再一方面提供了包含所述抗肿瘤坏死因子人源化抗体核 酸分子的重组载体， 包含所述核酸和 /或重组载体的宿主细胞， 以及所 述核酸、 载体和 /或宿主细胞的制备方法和 /或应用。
本发明的至少一种抗体具有至少一种活性， 例如但不限于中和 rhTNFa对 L929靶向细胞展现的毒性、 抑制和 /或竟争 TNF与受体和 / 或其他单抗例如但不限于阿达木单抗的结合。
本发明的再一方面提供了本发明的抗体和 /或组合物在抑制人 hTNFa活性的应用， 其中 hTNFa有关的疾病可以是例如脓毒症、 自身 免疫性疾病、 恶性肿瘤、 肺功能紊乱、 移植排斥、 细菌性脑膜炎、 脑 型疟、 AIDS和 AIDS相关综合症 (ARC)、 移植继发的巨细胞病毒感染。
本发明的再一方面提供了本发明的抗体和 /或组合物在制备用于 hTNFa的诊断性分析的药物中的应用， 其中所述抗肿瘤坏死因子人源 化单克隆抗体还可以被检测分子进行标记和 /或不进行标记 , 所述标记 包括放射性同位素； 荧光标记； 各种酶底物标记。 的应用， 其中所述分析方法包括竟争性结合分析、 直接或间接夹心分 析或免疫沉淀分析。
本发明的再一方面提供了至少一种组合物， 所述组合物包括本发 明的抗肿瘤坏死因子人源化抗体和 /或其编码核酸， 一种或多种可以选 自但不限于可药用载体、 赋形剂、 稀释剂、 添加剂等的辅剂。 所述组 合物可以任选地进一步包括至少一种其他抗体、 核酸、 辅剂或其任意 组合。
本发明的再一方面提供了包括预定量试剂和说明书的试剂盒， 所 述试剂包括本发明的抗体、 核酸和 /或组合物。 所述试剂盒还包括酶所 需的底物和辅因子。 所述试剂盒还可以进一步包括但不限于稳定剂、 緩冲剂等其他添加剂。 附图说明
本领域技术人员将理解下述附图仅是为了举例说明。 这些附图不 是要以任何方式限制本教导的范围。
图 1显示了抗体中和 TNFa杀伤 U937细胞作用曲线
图 2显示了 II型胶原诱导大鼠关节肿胀程度在体评分
图 3A显示了 Tgl97小鼠关节炎在体评分， 图 3B显示了治疗组对
Tgl97小鼠的的组织病理学评价， 图 3C显示了治疗组对 Tgl97小鼠的 关节炎评分 (AS)和组织病理 (HS)评分的影响。 具体实施方式
抗肿瘤坏死因子抗体
本发明的抗体包含由任意合适的多核苷酸编码的本文的抗体氨基 酸序列， 或任意分离的或制备的抗体。 人源化抗体或抗原结合片段优 选结合人肿瘤坏死因子， 并因此部分地、 基本上地或者全部地中和人 肿瘤坏死因子的至少一种生物活性，从而抑制由 TNF与 TNF受体相结 合所介导的信号传递过程以及生理过程。
本发明的人源化抗体可以是任意类型 (IgG、 IgA、 IgM、 IgE、 IgD 等)或同种型， 可以包含 κ或 λ轻链以及 α, μ, γ, ε或 δ重链。
本发明的至少一种抗体结合至少一种 TNF蛋白、 其亚基、 片段、 部分或其任意组合的至少一种特定表位。
本发明的至少一种抗肿瘤坏死因子人源化单克隆抗体具有以下氨 基酸序列，其中所述抗体的重链可变区的氨基酸序列如 SEQ ID NO: 1 , 并且所述抗体的轻链可变区的氨基酸序列如 SEQ ID NO: 2所示： SEQ ID NO: 1 :
QVQLVQSGPELKKPGASVKISCKASGYTFTHYGMHWVKQTPGRGL KWVGWINTYTGEPTYDADFQGRFTFSLETSVSTAFLQINSLKDEDL ATYFCARYDFDGFDYWGQGTTLTVS S SEQ ID NO: 2:
ENVLTQSPPILSASPGERVTMTCRASSSITFNYLHWYQQKSGDSPKV
WIYSTSNLVSGVPSRFSGSGSGTSYSLTISSLEAEDAATYYCQQYSD
YPYTFGGGTKLEIK
其中所述重链可变区的互补决定区 CDR-H1为 SEQ ID NO: 3所示 的氨基酸序列； CDR-H2为 SEQ ID NO: 4所示的氨基酸序列； CDR-H3 为 SEQ ID NO: 5所示的氨基酸序列； 其中构架区 FR-H1内氨基酸 16的 A能被 E替换， 氨基酸 17的 S能被 T替换， 氨基酸 20的 I能被 V替换 (SEQ ID NO: 11); FR-H2内氨基酸 3的 K能被 R替换， 氨基酸 9的 G能 被 S替换 (SEQ ID NO: 12); FR-H3内氨基酸 3的 T能被 V替换，氨基酸 7的 E能被 D替换， 氨基酸 10的 V能被 T替换， 氨基酸 14的 F能被 Y 替换，氨基酸 19的 S能被 T替换，氨基酸 27的 T能被 V替换 (SEQ ID NO: 13); 以及该人源化抗体的轻链可变区的互补决定区 CDR-L1为 SEQ ID NO: 6所示氨基酸序列； CDR-L2为 SEQ ID NO: 7所示氨基酸序列； CDR-L3为 SEQ ID NO: 8所示氨基酸序列，其中构架区 FR-L1内氨基酸 11的 L能被 M替换， 氨基酸 18的 R能被 E替换， 氨基酸 21的 M能被 I替换 (SEQ ID NO: 14); FR-L2内氨基酸 13的 W能被 L替换 (SEQ ID NO: 15); FR-L3内氨基酸 4的 S能被 A替换， 氨基酸 22的 L能被 V替换， 以及氨基酸 27的 A能被 F替换 (SEQ ID NO: 16)。
SEQ ID NO.3 :
HYGMH
SEQ ID NO: 4:
WINTYTGEPTYDADFQG
SEQ ID NO: 5:
YDFDGFDY
SEQ ID NO: 6:
RASSSITFNYLH SEQ ID NO: 7:
STSNLVS
SEQ ID NO: 8:
QQYSDYPYT
Figure imgf000007_0001
GTTLTVSS
Figure imgf000007_0002
ED(A/F)ATYYCQQYSDYPYTFGGGTKLEIK
SEQ ID NO: 11 :
^LVQSGPELKKl
SEQ ID NO: 12:
WV(K/R)QTPGR(S/G)LKWVG
SEQ ID NO: 13:
r/V)FSL(E/D)TS(
SEQ ID NO: 14:
ENVLTQSPPI(M/L)SASPGE(E/R)VT(M/I)TC
SEQ ID NO: 15:
WYQQKSGDSPKV(W/L)IY
SEQ ID NO: 16: 在本发明的一个实施方式中， 本发明的人源化抗体的重链恒定区 序列是人 IgGl的重链恒定区序列。
在本发明的一个实施方式中， 本发明的人源化抗体的轻链恒定区 序列是人抗体的轻链恒定区序列。
在本发明的一个优选实施方式中， 所述的抗肿瘤坏死因子人源化 单克隆抗体的氨基酸序列可通过插入、 缺失或保守性替换一个或多个 氨基酸残基， 优选为 1-3个氨基酸残基而被修饰。
通过一个或多个以任何组合形式存在的插入、 缺失或保守性替换 进行修饰的或变异的单克隆抗体在氨基酸序列上有所差异。 优选的变 异体中修饰是通过氨基酸保守性替换从上述本发明单克隆抗体中变化 而来。 所述保守性替换是指用性质相似的另一个氨基酸取代指定的氨 基酸。 下列以非限制性方式例举的氨基酸被认为是可进行保守性替换
(性质相似的）： a) 丙氨酸， 丝氨酸和苏氨酸； b)谷氨酸和天冬氨酸； c) 天冬酰胺和谷胺酰胺； d)精氨酸和赖氨酸； e) 异亮氨酸， 亮氨酸， 蛋氨酸和缬氨酸和 f) 苯丙氨酸， 酪氨酸和色氨酸。
根据本发明功能相当的单克隆抗体是一种变异体， 其中一个或多 个氨基酸残基，优选为 1-3个氨基酸残基被保守性替换。保守性替换是 指在芳香族氨基酸 Ala、 Val、 Leu和 lie之间一个替换另一个； 羟基残 基 Ser和 Thr之间的相互替换； 酸性残基 As 和 Glu之间的相互替换； 酰胺残基 Asn和 Gin之间的相互替换； 碱性残基 Lys和 Arg之间的相 互替换； 芳香族残基 Phe和 Tyr之间的相互替换。
另外， 本发明公开了与本文公开的氨基酸序列具有至少 50%同源 性的氨基酸序列， 或其片段和功能相当的氨基酸序列。 在一个实施方 式中， 该氨基酸序列与本文提供的氨基酸序列 SEQ ID NO: 1和 2具 有至少 75%的同源性，较优选为至少 85%的同源性，更优选为至少 90% 同源性， 更为优选的是至少 95%同源性， 更加优选至少 97%同源性， 最优选至少 99%的同源性。
各种形式的抗体被包括在本发明之中。 例如抗 hTNFa抗体可以是 全长的抗体 (例如具有完整的人 Fc区），或者抗体片段 (如 Fv, scFv, Fab, Fab，和 (Fab，)2)等。 此外， 抗体可以用可检测的标记物进行标记， 固定 在固相载体上， 和 /或偶联于异源化合物 (如细胞毒性物质）。
Fab是通过用蛋白酶 /木瓜蛋白酶处理 IgG抗体分子获得的。 这是 具有大约 50,000的分子量并具有抗原结合活性的抗体片段，其中在通过 木瓜蛋白酶 (在 H链的 224位切开氨基酸残基)获得的片段中， H链的大 约一半 N-端侧和整个 L链通过二硫键结合在一起。 本发明的 Fab也可 以通过将编码抗体的 Fab的 DNA插入到原核生物表达载体或真核生物 表达载体中以表达 Fab来生产。
Fab'是具有抗体结合活性的抗体片段，由 (Fab')2铰链区的二硫键切 开而产生，分子量大约 50,000,本发明的 Fab，也可以通过将编码抗体的 Fab'片段的 DNA插入到原核生物表达载体或真核生物表达载体中， 并 将载体导入到原核生物或真核生物中以表达 Fab，来生产。
(Fab')2是具有抗原结合活性的抗体片段， 分子量为大约 100,000. 其中在用蛋白酶 /胃蛋白酶 (在第 234个氨基酸残基处切开 H链)处理 IgG 抗体而获得的片段中， Fab通过铰链区中的二硫键连接在一起的抗体片 段略大。 本发明的 (Fab，)2可以通过用胃蛋白酶处理抗体来生产。 此外， 本发明的 (Fab，)2也可以通过用硫醚键或二硫键连接 Fab，来生产。
scFv是具有抗原结合活性的抗体片段， 是使用适当的肽接头将一 条链 VH和一条链 VL连接在一起。 本发明的 scFv的生产可以通过获得 编码抗体的 VH和 V] cDNA,构建编码 scFv的 DNA, 将编码抗体的 scFv的 DNA插入到原核生物表达载体或真核生物表达载体中 ,然后将 表达载体导入原核生物或真核生物中表达 scFv。
核酸
本发明的核酸编码如 SEQ ID NO: 1-16中的至少一种、 其特定片 段、变体或共有序列的至少 70-100 %的连续氨基酸的核苷酸序列，可以 根据本文所描述的或本领域公知的方法获得编码至少一种抗 TNF抗体 的本发明的核酸分子。
本发明的核酸分子可以是 RNA形式，例如 mRNA、 hnRNA、 tRNA 或任意其它形式， 或可以是 DNA形式， 包括， 但不限于通过克隆或合 成产生， 或其任意组合而产生的 cDNA和基因组 DNA。 DNA可以是 双链或单链。 DNA或 RNA的至少一条链的任意部分可以是编码链， 也称作有义链， 或可以是非编码链， 也称作反义链。
如本文中所指出的， 包含编码抗 TNF抗体的核酸的本发明的核酸 分子可以包括， 但不限于自身编码抗体片段的氨基酸序列的核酸； 完 整抗体或其部分的编码序列； 抗体、 片段或部分的编码序列， 以及其 它的序列， 例如至少一种信号前导肽或融合肽的编码序列， 它具有或 不具有前面提到的其它编码序列， 如至少一种内含子， 同时具有其它 的非编码序列， 包括， 但不限于非编码 5'和 3'序列， 如在转录、 mRNA 加工，包括剪接和聚腺昔酸化信号中起作用（例如 mRNA的核糖体结合 和稳定性)的转录的、 未翻译的序列； 编码其它氨基酸， 如提供其它功 能的氨基酸的其它序列。 因此， 编码抗体的序列可以与标记序列融合， 例如与促进包含抗体片段或部分的融合抗体纯化的肽的编码序列融 合。 本发明的核酸还包括选择性与此处描述的多核苷酸杂交的多核苷 酸。
本发明的核酸可以利用本领域公知的 (a)重组方法， （b)合成技术， (c)纯化技术或其组合进行制备。
本发明的核酸如 DNA、 RNA、 cDNA、基因组 DNA或其任意组合 可以用本领域技术人员公知的任何克隆方法从生物来源中获得。 在一 些实施方案中， 在严格条件下选择性与本发明的多核苷酸杂交的寡核 苷酸探针被用于鉴定 cDNA或基因组 DNA文库中的所需序列。 RNA 的分离和 cDNA 以及基因组文库的构建是本领域普通技术人员所公知 的。
可以采用基于本发明的多核苷酸序列的探针筛选 cDNA或基因组 文库。可以用探针与基因组 DNA或 cDNA序列杂交， 以分离相同或不 同生物体中的同源基因。 本领域技术人员将理解， 在测定中可以使用 不同严格性程度的杂交， 杂交或洗涤介质中的任意一个都可以是严格 的。 当杂交条件更严格时， 探针和靶序列之间的互补性必须更高， 这 样才能形成双链体。 严格性程度可以由温度、 离子强度、 pH和曱酞胺 等部分变性溶剂的存在中的一个或多个条件进行控制。 例如， 杂交的 极性可以通过例如在 0 % -50 %的范围内操作曱酞胺的浓度而改变。可检 测的结合所要求的互补性程度 (序列等同性)将根据杂交介质和 /或洗 涤介质的严格性而改变。 互补性程度最佳为 100 %或 70-100 % , 或其中 的任意范围或任意值。 然而， 应该理解， 探针和引物中的小量序列变 异可以通过减少杂交和 /或洗涤介质的严格性而补偿。
扩增 RNA或 DNA的方法是本领域公知的。 已知的扩增 RNA或 DNA 的方法包括， 但不限于聚合酶链式反应 (PCR)和相关的扩增方法 和 RNA介导的扩增。
本发明的核酸也可以根据已知方法通过直接化学合成而制备。 化 学合成一般产生单链核苷酸， 该单链核苷酸可以通过与互补序列杂交 或通过用单链作为模板， 采用 DNA 聚合酶进行聚合而转化为双链 DNA。 人源化单克隆抗体表达载体的构建
将含有人源化抗体重链可变区序列 VH基因片段的质粒 (pHu-VH)作 为模板，使用 5'引物 FVHX (5，-CGCGCAAG-CTTCCTCGAG-3， SEQ ID NO: 17)和 3'引物 RVCG (5 ' CGATGGGCCCTTGGTGGA3 ' SEQ ID NO: 18)得到 5'片段， 含有人源化抗体的重链可变区 (VH)和人 01重链恒定 区 (C ) 5'端的 7个氨基酸的基因。在此同时， 用从人白细胞制备的 RNA 以合适的 5'引物 HuCGF (5 ' -ACCAAGGGCCCATCGGTCTTC-3 '； SEQ ID NO: 19) 和 3' 引 物 HUCGE (5 ' -CGGAATTCTCATTTACCC GGAGACAGGGA 3，， SEQ ID NO: 20) 通过逆转录反应和 PCR得到含 人 IgGi重链恒定区 (C ) 编码序列的基因。 最后， 将含有人源化抗体的 重链可变区 (VH)的片段与以上的人 基因通过 PCR,用 5'引物 (FVHX, SEQ ID NO: 17)和 3'引物 (HUCGE, SEQ ID NO: 20), 将人源化抗体的 重链可变区 (vH)和人 c 基因片段连接起来， 得到含有重链编码序列、 长度约为 1400 b 的基因片段。 此基因片段以内切酶 Hind III和 EcoRl 处理后， 插入载体如 PUC19(ref: Yanisch-Perron , C , Vieira, J. and Messing, J. (1985) Gene, 33 , 103-119.)中。
将含有人源化抗体轻链可变区序列 VL基因片段的质粒 (pHu-VL)作 为模板，使用 5'引物 FVHX (SEQ ID NO: 17)和 3'引物 VKCKO (5' -AGA
SEQ ID NO: 21)得到 5'片段，含有人源化抗体的轻链可变区 (VL) 和人 κ 轻链恒定区 (CK) 5'端的 7个氨基酸的基因。在此同时， 用从人白细胞制 备的 RNA以合适的 5'引物 HuCKF (5' -GTG GCT GCA CCA TCT GTC TTC -3， SEQ ID NO: 22)和 3'引物 HUCKB (5' -TGC GGA TCC CTA ACA CTC TCC CCT GTT GAA -3，， SEQ ID NO: 23) 通过逆转录和 PCR 得到含人 κ轻链恒定区 (CK) 编码序列的基因。 最后， 将含有人源化抗 体的轻链可变区 (VL)的片段与以上的人 CK基因通过 PCR, 用 5'引物 (FVHX, SEQ ID NO: 17)和 3'引物 (HUCKB , SEQ ID NO: 23), 将人源 化的轻链可变区 (VL)和人 CK基因片段连接起来， 得到含有轻链编码序 列、长度约为 700 b 的基因片段。此基因片段以内切酶 Hind III和 Bam HI处理后， 插入载体如 PUC19(ref: Yanisch-Perron, C.， Vieira, J. and Messing, J. (1985) Gene, 33 , 103- 119.)中。
用上述方法中获得的重链或轻链的编码 cDNA、或编码其修改产物 的 cDNA插入到 pcDNA3 (购自 Invitrogen USA, Carlsbad, CA USA) 载 体中， 构建 pHu— anti-TNFa人源化表达载体。 该表达载体质粒含有在 哺乳动物细胞中高水平表达所需的巨细胞病毒早期基因启动因子-增强 子。 同时， 载体质粒中含有可选择标记基因， 从而在细菌中赋予氨苄 青霉素抗性， 而在哺乳动物细胞中赋予 G418抗性。 另外， 载体质粒中 含有 DHFR基因， 在合适的宿主细胞中， 能以氨曱喋呤 (Methotrexate , MTX, Sigma)共扩增嵌合抗体基因和 DHFR基因（参见， 例如 Axel , R.,等人的美国专利 5,179,017号； Kaufman,R.和 Sharp,R , J.Mol. Biol. 159:601-621 ,1982)。 抗体宿主细胞
本发明也涉及用重组载体基因工程化的宿主细胞， 以及通过本领 域公知的重组技术产生至少一种抗 TNF抗体。
多核苷酸可以任选与含有可选择标记的载体连接， 用于在宿主中 增殖。 一般地， 将质粒载体导入一种沉淀物， 例如磷酸钙沉淀， 或导 入具有带电脂质的复合物。
宿主细胞的适当培养基和培养条件是本领域公知的。 适当的载体 是本领域技术人员容易理解的。 可以通过磷酸钙转染、 DEAE —右旋 糖昔介导的转染、 阴离子脂质介导的转染、 电穿孔、 转导、 感染或其 它已知方法将载体构建体导入宿主细胞。
本发明的抗肿瘤坏死因子人源化单克隆抗体的宿主细胞由中国仓 鼠卵巢细胞 (Chinese Hamster Ovary)衍生而得， 经包含抗 -TNFa基因内 码的质粒转殖， 及随后一系列严谨、 特定的筛选过程获得， 其间包括 药物筛选、 基因放大和单细胞克隆以建立最终的细胞株。 本发明的宿 主细胞中国仓鼠卵巢细胞株 CHO HUAT 132于 2011年 3月 7日保藏于中 国典型培养物保藏中心， 保藏编号 CCTCC NO. : C201117。
本细胞株的细胞能在无血清培养液中悬浮繁殖； 在 2升发酵罐中培 养时， 于 16-20天培养周期结束后其分泌于培养液中抗 -TNFa的水平将 不低于每升一克 (1 g/L)。本细胞株所生产的抗 -TNFa为人源化单克隆抗 体。 与 TNFa结合活性
本发明的抗肿瘤坏死因子人源化单克隆抗体仅与重组人源肿瘤坏 死因子 (rhTNFa, 标靶分子)有特定亲和力， 不与其他任何蛋白分子交 叉结合。 在对其标靶分子亲和力的竟争性测试中， 可测得与阿达木单 抗 (阿达木单抗)相近的亲和力。
本发明的抗肿瘤坏死因子人源化单克隆抗体还具有活性可以中和 rhTNFa对 L929靶向细胞展现的毒性， 其 EC50值与阿达木单抗 的 EC50值相似， 在 20.4 ng/mL至 50 ng/mL浓度范围内。 治疗用途
抗 hTNFa抗体可用于治疗和预防 TNFa有关的疾病， 其中 hTNFa 有关的疾病可以是例如脓毒症、 自身免疫性疾病、 恶性肿瘤、 肺功能 紊乱、 移植排斥、 细菌性脑膜炎、 脑型疟、 AIDS和 AIDS相关综合症 (ARC),移植继发的巨细胞病毒感染， 以下进一步讨论本发明的抗体和 抗体部分在治疗 hTNFa有关疾病的用途
1)脓毒症
肿瘤坏死因子在脓毒症病理学中已有确定地位， 其生物学效应包 括低血压、 心肌抑制、 血管漏出综合症、 器官坏死等 (参见例如美国专 利 5,231,024号），因此本发明的人源化抗体和抗体部分可用于治疗任何 临床背景中得脓毒症， 包括脓毒症性休克、 内毒素性休克、 革兰氏阴 性脓毒症和毒性休克综合症。
2) 自身免疫性疾病 用， 例如， 已发现 TNFa参与激活组织炎症并引起类风湿性关节炎中 的关节破坏（参见例如美国专利 5,231,024 号； Moeller,A.等 (1990)Cytokine 2:162-169;), 也发现 TNFa在糖尿病中参与促进胰岛细 胞死亡和介导对少突神经胶质细胞的细胞毒和诱导炎症斑。
本发明的人源化抗体和抗体部分可以用于治疗自身免疫疾病， 特 别是那些与炎症有关的自身免疫疾病， 包括类风湿性关节炎、 类风湿 性脊髓炎、 骨关节炎和痛风性关节炎、 变态反应、 多发性硬化症、 自 身免疫性糖尿病、 自身免疫性眼色素层炎和肾病综合症。
3) 恶性肿瘤
已发现肿瘤坏死因子在恶性肿瘤中参与诱导恶病质、 刺激肿瘤生 长、 增强转移潜力和介导细胞毒性。 因此， 可以使用本发明的抗体和 抗体部分治疗恶性肿瘤， 抑制肿瘤生长或转移和 /或减轻恶性肿瘤继发 的恶病质。 可以将该抗体或抗体部分系统性给药或局部给予该肿瘤部 位。
4)肺功能紊乱
已知肿瘤坏死因子参与成人呼吸窘迫综合症 (ARDS)的病理生理 学， 包括刺激白细胞-内皮细胞激活、 细胞毒性导向肺细胞和诱导血管 漏出综合症。 因此， 可以用本发明的抗体和抗体部分治疗肺功能紊乱， 包括成人呼吸窘迫综合症、 慢性肺炎、 肺结节病、 肺纤维化和硅肺。
5) 肠功能紊乱
可以使用本发明的人抗体和抗体部分治疗肠功能紊乱， 例如特发 性炎症性肠病， 该病包括两种综合症， 即局限性回肠炎和溃疡性结肠 炎。
6) 移植
已发现肿瘤坏死因子可能是同种移植排斥和移植植物抗宿主病 (GVHD)的关键介质并参与介导在用导向 T细胞受体 CD3复合物的大 鼠抗体 OKT3 抑制肾移植排斥时观察到的副反应（参见例如 Suthanthiran,M.和 Strom,T.B.(1994) New Engl,J.Med.331 :365-375) 因此， 可以用本发明的抗体和抗体部分抑制移植排斥， 包括同种移植 和异种移植排斥和抑制 GVHD.
7)传染病
可以用本发明的抗体和抗体部分治疗传染病， 包括细菌性脑膜炎、 脑型疟、 AIDS和 AIDS相关综合症 (ARC)以及移植继发得巨细胞病毒 感染， 也可以用于减轻与传染病有关的症状， 包括因感染 (例如流感) 引起的发烧和肌痛和感染继发的恶病质 (例如 AIDS或 ARC继发的：)。 分析及诊断用途
本发明抗体可以用在任何一种已知的分析方法中， 例如竟争性结 合分析， 直接或间接夹心分析和免疫沉淀分析。 Zola, 《单克隆抗体： 技术手册》 (Monoclone Antibodies; A Manual of Techniques) , pp. 147-158(CRC Press, Inc. , 1987)。
竟争性结合分析依赖于标记过的标准物与被测样品中分析物竟争 结合有限量抗体的能力。 被测样品中 hTNFa的量与抗体结合的标准物 的量成反比。 为了方便测定被结合标准物的量， 通常令抗体在竟争前 或竟争后不溶解， 这样就可以方便地将与抗体结合的标准物和分析物 与未结合的标准物和分析物分离。
夹心分析法涉及使用两种抗体， 各自结合待测蛋白不同的免疫原 性部位或表位。 在夹心分析中， 被测样品分析物与固定在固相载体上 的第一抗体结合， 然后第二抗体与分析物结合， 由此形成不溶性三部 分复合物。 参见美国专利 4,376,110。 第二抗体本身可以是用可检测部 分标记过的 (直接夹心分析法），或者利用被可检测部分标记的抗免疫球 蛋白抗体来测定（间接夹心分析法）。 例如， 夹心分析法之一是 ELISA, 其中的可检测部分是酶。
抗 hTNFa抗体还可用于 hTNFa的诊断性分析中，例如检测其在特 定细胞、 组织或血清中的表达。 这种诊断方法可用于诊断自身免疫疾 病等的病因。
抗体通常用可检测分子进行标记。 有许多标记可以使用， 它们可 以大致如下分类：
(a)放射性同位素， 例如 l llln、 99Tc、 14C、 1311、 1251、 3H、 32P 或 35S。 可以利用例如 《现代免疫学方法》 （Current Protocols in Immunology)第 1和第 2卷, Coligen等编 , Wiley-Interscience , New York, New York, Pubs.(1991)中所述的方法以放射性同位素来标记抗体， 放 射性可以利用闪烁计数法来测定,以利用免疫闪烁照相术来定位患病部 位。
(b) 荧光标记， 例如稀土螯合剂 (铕螯合剂）或萤光素及其衍生物， 罗丹明及其衍生物， 丹酰， 丽丝胺， 藻红素和德克萨斯红 (Texas red)。 荧光标记可以利用例如上文《现代免疫学方法》 中所述的方法与抗体 偶联。 荧光可以利用荧光计来定量。
(c)有各种酶底物标记可供使用， 而美国专利 4,275,149公开了其 变。 例如， 酶催化底物的颜色变化， 这种变化可以用分光光度计来测 定， 或者， 酶改变底物的荧光性或化学发光性。 前文已经说明了定量 测定荧光变化的技术。 化学发光底物因化学反应而被电激发， 并由此 发光， 发出的光可以被测定 (例如利用化学光度计)或向荧光受体供能。 酶标记包括例如荧光素酶 (例如萤火虫萤光素酶和细菌萤光素酶； 美国 专 利 4,737,456) , 萤 光 素 ， 2,3- 二 氢 二 氮 杂 萘 二 酮 (2,3-dihydrophthalazinediones).苹果酸脱氢酶，脲酶、过氧化物酶如辣根 过氧化物酶 (HRPO), 碱性磷酸酶、 b-半乳糖苷酶， 葡糖淀粉酶、 溶菌 酶、 糖氧化酶 (例如葡萄糖氧化酶， 半乳糖氧化酶和葡萄糖 -6-磷酸脱氢 酶）， 杂环氧化酶 (例如尿酸酶和黄嘌呤氧化酶)， 乳过氧化物酶， 过 氧化物酶等。 0， Sullivan等在"制备用于酶免疫分析的酶-抗体偶联物的 方法，， , 《酶学方法》 ("Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay" (Methods in Enzym.) (J. Langone 和 H. Van Vunakis 编 ) , Academic press , New York , 73: 147-166(1981)中描述了酶与抗体偶联的技术。
酶 -底物的组合物包括， 例如：
(i)辣根过氧化物酶 (HRP)和作为底物的氢过氧化物酶， 其中的氢 过氧化物酶使染料前体 (例如邻苯二胺 (OPD)或盐酸 3 , 3' , 5 , 5'- 四曱 基联苯胺 (TMB))氧化；
(ii)碱性磷酸酶 (AP)和作为生色底物的对硝基苯磷酸；
(iii) b-D-半乳酸酐酶 (b-D-Gal)和生色底物 (例如对硝基苯 -b-D-半乳 糖苷酶)或荧光底物 4-曱基伞形酮 -b-D-半乳糖苷酶。
对本领域技术人员来说还有许多其它酶 -底物组合。 对这些组合的 综述可参见美国专利 4275149和 4318980。有时， 标记物与抗体间接偶 联。 技术人员也知道各种获得所述组合物的方法。 例如， 抗体可以与 生物素 (biotin)偶联， 上述三大类标记中的任何一种都可以与亲和素偶 联， 或者正相反。 生物素选择性地结合亲和素， 标记可以间接方式与 抗体偶联。 或者， 为了将标记与抗体间接偶联， 抗体可以与小的半抗 原（例如地高辛)偶联， 而上述不同类型的标记之一与抗半抗原抗体 (例 如抗地高辛抗体)偶联。 这样就获得的标记与抗体的间接偶联。
在本发明另一实施方案中， 抗 hTNFa抗体不必被标记， 其存在可 以利用标记过的结合该 hTNFa抗体的抗体来检测。 亲和纯化试剂
本发明的抗体可以用作亲和纯化试剂。 在这种方法中， 抗体利用 本领域公知的方法固定在例如 Sephadex树脂或滤纸的固相上。被固定 的抗体与待纯化的含 hTNFa的样品接触，然后用合适的溶剂洗涤载体， 所述的溶剂能够基本上去除样品中除了与固定化抗体结合的 hTNFa之 外所有其它物质。 药用组合物和给药方式
可以将本发明的抗体和抗体部分加入适于给予受治疗者的药物组 合物中， 其中， 该药物组合物包括本发明抗体和可药用赋形剂， 药用 赋形剂包括任何所有的生理适用的溶剂、 分散介质、 抗菌剂、 抗真菌 剂、 等渗剂、 包衣、 吸收延迟剂等等。 本发明的药物组合物可以有各 种形式， 包括例如液半固体和固体剂量形式。
可将在药学上可接受的剂型中的本发明抗—hTNFa抗体， 用已知方 法施用于人。 所述方法包括在静脉内（例如静脉注射浓缩药团 (bolus)或 在一段时间内连续输注）、 肌内、 腹膜内、 脑脊髓腔内、 皮下、 动脉内、 滑模腔内、 鞘内注射、 口服、 局部或吸入途经使用。 抗体还可合适地 通过瘤内、 瘤周围、 损伤部位内、 损伤部位周围的途经给药， 以发挥 局部和全身的治疗效果。 腹膜内途经预计特别有用， 例如对于治疗卵 巢癌。
为了预防或治疗疾病来说， 抗体的合适剂量将取决于上述定义的 待治疾病的类型、 疾病的严重程度和病程、 抗体给予是用来预防还是 用来治疗、 以前的治疗情况、 患者病史和对抗体的应答性， 以及主治 医师的独立判断。 抗体适合一次性或系列地给患者使用。
根据疾病的类型和严重程度， 不论是一次或多次分开给药， 还是 连续输注。 1微克 /公斤至 50毫克 /公斤 (例如 0.1-20毫克 /公斤）的抗体是 给患者使用的最初候选剂量。 典型的日剂量或周剂量可在约 1毫克 /公 斤 -20毫克 /公斤或以上，这取决于上述提到的因素。对于几天或更长时 间内的重复给药 (这取决于病况)来说，治疗需持续直至疾病症状发生所 期望的抑制。 但是， 也可以使用其它给药方案。 所述治疗的进展可以 方便地利用常规技术和分析方法来监测。 产品
1) 注射剂
本发明另一实施方案提供了一种含有用于治疗上述疾病的材料的 产品。 该产品包括容器和标签。 合适的容器包括例如普通瓶、 药瓶、 注射器和试管。 容器可以用各种材料制成， 例如玻璃或塑料。 容器中 含有治疗疾病的有效组合物， 并具有一个无菌的出入口（例如容器可以 是一个有塞子的静脉输液袋或药瓶， 该塞子可用皮下注射针头穿透)。 该组合物中的有效成分是抗 hTNFa抗体。 容器上或与容器相联的标签 说明组合物所治疗的特定病症。 产品还可以含有另一个容器， 其中包 含药学上可接受的緩冲液， 例如磷酸盐緩冲液、 林格式 (Ringer)溶液和 葡萄糖溶液。 根据商业上的需要或使用者的需要， 它还可以包括其他 材料， 例如其他緩冲液、 稀释剂、 滤器、 针头、 注射器、 以及附有使 用说明的包装说明书。
2)緩释制剂
本发明的抗肿瘤坏死因子人源化单克隆抗体可以用于制备緩释制 剂。 合适的緩释制剂包括例如包含所述抗体的固体疏水聚合物的半渗 透基质体， 所述的基质体是有形的物体， 例如膜或微胶嚢。 合适的緩 释基质体包括例如聚酯、 水凝胶 (例如聚 (曱基丙烯酸 2-羟基乙酯)或聚 乙烯醇)、 聚交酯 (美国专利 3 , 773 , 919)、 L-谷氨酸和 L-谷氨酸乙酯 的共聚物、 非降解性乙烯乙酸乙烯酯、 诸如 Lupron DepotTM (由乳酸- 乙醇酸共聚物和 leuprolide acetate组成的可注射微球)之类可降解乳酸- 乙醇酸共聚物和聚 -D-(-)-3-羟基丁酸。 诸如乙烯乙酸乙烯酯和乳酸-乙 醇酸之类聚合物能够使分子译放持续 100天以上， 有些水凝胶可在较 短的时间内译放蛋白质。 当胶嚢化的抗体在体内保留较长时间时， 它 们可能会因为在 37°C接触水分而变性或凝聚， 结果造成生物活性降低 并可能造成免疫原性改变。 根据有关的机制可以设计出合理的稳定化 策略。 例如， 如果发现凝聚机制是通过硫-二硫键互换反应而形成了分 子间的 S-S键， 稳定化可以是通过修饰巯基残基、 冻干酸性溶液、 控 制含水量、 使用合适的添加剂和设计特殊的聚合基质组合物而达到。 试剂盒
为了方便， 本发明抗体可以试剂盒的形式提供， 即将预定量的试 剂与进行诊断分析的说明书的包装组合在一起。 如果抗体是用酶标记 的， 试剂盒将包含酶所需的底物和辅因子 (；例如提供可测定的生色团和 荧光团的底物前体)。 此外， 还可能包括其他添加剂， 例如稳定剂、 緩 冲剂 (例如封闭緩冲剂或裂解緩冲剂)等。为了使所提供的试剂浓度能使 得分析的灵敏度最高， 各种试剂的相对量变化很大。 具体地说， 试剂 可以是干粉， 通常是冻干粉末形式的， 可包括赋形剂在内， 它们一经 溶解将形成具有合适浓度的试剂溶液。
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例 仅用于说明本发明而不用于限制本发明的范围。 下列实施例中未注明 具体条件的实验方法， 通常按照常规条件， 例如 Sambrook等人， 分子 克隆：实马 室手册 (New York; Cold Spring Harbor Laboratory Press, 1989) 中所述的条件， 或按照制造厂商所建议的条件。 实施例
实施例 1: 抗 hTNFa鼠单克隆抗体的生产
1)免疫
将重组 hTNFa(rhTNFa, 购自 Pepro Tech Inc.)对一些 7至 11周龄 的雌性 Balb/c小鼠进行 IP或 ID免疫， 这些重组人 TNFa采用等体积 的 TITERMAX或弗氏完全佐剂进行乳化， 其最终体积为 100-400 μΙ^。 每只小鼠在 1-7,5-12,10-18,17-25和 /或 21-34天后用等体积 TITERMAX 或弗氏不完全佐剂乳化的 TNF进行 IP(l-400 g)和 SC(1400 gx2)免疫。 12-25 和 25-40 天之后可以通过不抗凝条件下的框后穿刺而从小鼠取 血。 然后使血液在 RT下凝固 1小时， 收集血清， 根据已知的方法采用 TNFa ELA测定滴度。 当重复注射不导致滴度增加时进行融合。 此时， 可以用稀译于 100 μΐ^生理盐水的 1-400 μg TNFa给予小鼠最后一次加 强注射。 3天后， 通过断颈推处死小鼠， 在无菌条件下取出脾， 浸入含 有 1000 U/mL青霉素、 100 g/mL链霉素、 和 0.25 g/mL两性霉素 B(PSA)的 10 mL冰冷盐酸緩冲盐水 (PBS)。 通过用 PSA-PBS无菌灌注 脾而收获脾细胞， 用台盼蓝染色排除法计数， 并且重悬于含 25 mM Hepes的 RPMI 1640培养基。
2) 小鼠血清测试
用 PBS 中 的 2 g/mL TNFa 包被平板过夜。 在含有 0.02%(v/v)Tween20的 0.15 M盐水中洗涤后，用 PBS中的 l%(w/v)BSA, 以 200 μΐ^ /孔在 RT下封闭各个孔 1小时。立即使用平板或在 -20°C下冷 冻， 以便将来使用。在 TNFa包被的板上以 50 μL /孔在 RT下孵育小鼠 血清稀译液 1小时。 洗涤平板， 然后用 1% BSA-PBS中 1:30000特异 性稀释的 50 μΙ /^Ι HRP标记的 IgG Fc在 RT下探测 1小时，再次洗涤 平板，在 RT下加入 100 L /孔柠檬酸盐-磷酸盐底物溶液 (0.1 M柠檬酸 和 0.2 M硫酸钠， 0.01% ¾02和 1 mg/mL OPD)15分钟，然后加入 25 /孔终止溶液 (4Ν硫酸)，在 490 nm下通过自动平板分光光度计读出 OD 值。
3) 细 融合
将血清测试鉴定出的， 具有高水平抗 hTNFa抗体血清的小鼠存活 脾细胞与小鼠骨髓瘤细胞融合， 两者比例为 1:1至 10:1。作为非限制性 的实例， 将脾细胞和骨髓瘤细胞一起沉淀， 然后在 37°C下， 在 1 mL 50%(w/v)PEG/PBS溶液 (PEG分子量 1450, sigma)中重悬沉淀 30秒以 上。 然后通过緩慢加入 10.5 mL含有 25 mM Hepes(37°C)的 RPMI 1640 培养基 1分钟以上， 以便终止融合。 以 500-1500 rpm将融合细胞离心 5分钟。然后将细胞重悬于 HAT培养基 (RPMI 1640培养基，含有 25 mM Hepes, 10% Fetal Clone I血清 (Hyclone),l mM丙酮酸钠， 4 mM L-谷氨 酰胺， 10 g/mL庆大霉素， 2.5% Origen培养补充物 (Fisher), 10% 653 调节的 RPMI 1640/Hepes培养基， 50 μΜ 2-巯基乙醇， 100 μΜ次黄嘌 呤和 16 μΜ胸苷）， 并且以 200 μΙ7孔平铺于 15个 96孔平底组织培养 板。然后将板置于含有 5% C02和 95%空气的潮湿的 37°C孵育器中 7-10 天。 实施例 2: 抗 hTNFa鼠源抗体的定性
抗 hTNFa抗体的定性有两种测定法。 一种方法是测定抗体对阿达 木单抗与 hTNFa的竟争结合， 另一种方法是测定抗体在 L929细胞毒 性测定中中和 hTNFa的能力。 下文中分别对这两种方法及其实验结果 进行了描述。
1) 与阿达木单抗的竟争结合测定
以辣根过氧化物酶 (HRP)标记抗 hTNFa人抗体阿达木单抗作为试 剂。 将 rhTNF(5(^l, 0.05 g/mL)包被酶标板,室温过夜。 弃去包被溶液， 用溶解在磷酸盐緩冲盐水 (PBS)的 1%脱脂奶封闭各孔 0.5小时，用含有 0.05% 吐温 (Tween)20的 PBS洗孔。 然后每孔加入 50 μΐ生长培养基与 50 μΐ HRP标记的阿达木单抗的混合液。 以未标记的阿达木单抗和不含 抗体的培养基分别作为阳性与阴性对照。 以此方法可筛选出含有高能 力抑制 HRP标记阿达木单抗与 rhTNfa结合的鼠单克隆抗体。 将含有 抑制 HRP标记阿达木单抗与 rhTNFa结合的孔放大并亚克隆， 继续进 一步分析数个表现出此抑制作用的鼠单克隆抗体， 最后筛选出二杂交 瘤细胞。 将此二杂交瘤细胞扩大培养， 取澄清液提纯， 得鼠单克隆抗 体 TM2-11-12 与 TM2-6-3。 以纯化的鼠单克隆抗体 TM2-11-12 与 TM2-6-3进行竟争结合测定， 鼠抗体 TM2-6-3在高达 1 g/mL浓度时, 只能竟争掉约 50% 阿达木单抗与 hTNFa 的结合。 另一个鼠抗体 TM2-11-12 则显示与未标记的阿达木单抗具有同等优良的竟争能力， 在大约 0.05 g/mL浓度时 (相当于 3x10 ° M),即可竟争掉约 50% 阿达 木单抗与 hTNf^的结合。
2) 抗 hTNFa鼠源抗体的定性： 体外中和 hTNFa的活性测定 抗 hTNFa 鼠源抗体与嵌合抗体中和 hTNFa 的生物活性皆可用 L929 细胞毒性测定， 方法如下所述。 将 96 孔培养板的每孔注入共 7.5xl03 L929细胞 (ATCC) (105 / mL, 75 μΐ), 置于 37°C 5% C02的培养 箱内 24 小时。 L929 细胞的生长培养基为含有 5%胎牛血清的 RPMI-1640(GIBCO)„ 用另一块 96孔培养板将含有抗 hTNFa抗体的溶 液用 RPMI生长培养基双份进行 1/2连续稀释， 并加入 rhTNFa使每个 样品孔 rhTNFa的最终浓度为 5 ng/mL,将有混合液的培养板放在 37 °C , 5% C02的培养箱内 2小时后， 再将含有抗体与 rhTNFa的混合液加入 L929细胞孔内，每行各孔抗体浓度依次为 0.001至 2 g/mL。将此培养 板置入 37°C , 5% C02。 3天后测存活细胞时， 加入 20 μΐ PBS中有 2.5 mg/mL 溴化 3(4,4-二曱基噻唑 -2-基) 2,5-二苯基-四唑翁 (MTT; 购自 Sigma Biochemicals)在 37°C培育 4小时， 再加入 100 μΐ 0.01 N HC1 中 有 10%十二烷基硫酸钠 (SDS)过夜。 其后测定每孔于 540/690 nm光密 度。 将光密度度数与抗体浓度绘制曲线。 分析结合曲线得 IC5。， 即在 此抗体浓度时， 50%的 rhTNFa对 L929细胞的毒性可被中和。 因此可 用 IC5。值来比较个抗体抑制 hTNFa细胞毒性的能力。 数个抗 hTNFa 鼠源抗体 (TM2-11-12, TM2- 10-20, TM2-2-2)的 IC5。值与阿达木单抗的 IC5。值皆在 0.01至 0.04 g/mL浓度范围内， 即皆具有相似的能力中和 rhTNFa引起的 L929细胞毒性。抗 hTNFa鼠源抗体 TM2- 11-12被选作 进一步制备嵌合抗体。 实施例 3: 克隆 TM2-11-12鼠源抗体的重链和轻链
1) TM2-11-12鼠源抗体的重链可变区的克隆
为设计鼠源抗体的人源化， 首先须获得含鼠源抗体 TM2-11-12重 链和轻链可变区编码序列的 DNA片段。用 RNA纯化试剂盒 (Invitrogen Corp.)从 TM2-11-12 小鼠杂交瘤细胞分离出 RNA , 以此制备 cDNA(GeneRacer试剂盒， Invitrogen Corp.)。 通过聚合酶链反应 (PCR) 使用 5，引物 (5，- CGACTGGAGCACGAGGACACTGA-3 ' , SEQ ID NO:
24)和 3，引物(5，-丁(^入00000^入0丁00入丁入0入0入0入-3，， SEQ ID NO:
25)从 cDNA中分离重链可变区 DNA片段， 3，引物与小鼠 IgGl重链恒 定区同源反义。 将这些得到的 DNA 片段克隆进 TOPO TA 载体 (Invitrogen)并测序。 重链可变区的氨基酸序列（SEQ ID NO: 34), 互补 决定区的氨基酸残基为 CDR-H1(SEQ ID NO: 3), CDR-H2(SEQ ID NO: 4)和 CDR-H3(SEQ ID NO: 5)。 互补决定区的定义参见 Kabat E.等人的 Sequences of Proteins of Immunological Interest第 5版 U.S. Department of Health and Human Services,NIH Publication No.91-3242。
2) TM2- 11 - 12鼠源抗体的轻链可变区的克隆
以类似重链可变区克隆的 PCR方法， 使用 SEQ ID NO: 29作为 5， 引物和另一个与小鼠免疫球蛋白 K 轻链恒定区同源反义的 3'引物
(5 ' -CACTGGATGGTGGGAAGATGGATA-3 ' , SEQ ID NO: 26),从 cDNA 中分离轻链可变区 DNA片段。 将这些得到的 DNA片段克隆进 TOPO TA载体并测序， 发现有两类克隆。 约 3/4的克隆显示部分核苷酸序列 不能翻译成能够通读的氨基酸序列（未显示其序列）。此类克隆为畸变轻 链信使 RNA, 不能编码有功能的抗体轻链蛋白。 另外约 1/4的克隆显 示的核苷酸序列则可完全翻译为能够通读的氨基酸序列， 此类克隆衍 生于有功能的轻链信使 RNA。将这些得到的 DNA片段克隆进 TOPO TA 载体 (Invitrogen)并测序。 轻链可变区的氨基酸序列（SEQ ID NO: 35) , 互补决定区的氨基酸残基为 CDR-L1(SEQ ID NO: 6), CDR-L2(SEQ ID NO: 7)和 CDR-L3(SEQ ID NO: 8)。 此氨基酸序列用于轻链的人源化设 计。 实施例 4: 重链与轻链可变区的人源化设计
为了保留抗原结合的活性， 在人源化过程中所有轻链和重链高变 区内的氨基酸残基仍然沿用 TM2-11-12鼠源抗体的序列。 人源化设计 则是依照人抗体的序列改变构架区内的氨基酸残基， 设计了具有各种 不同修饰的人源化抗体的重链可变区和轻链可变区， 通过计算机模拟 技术， 将抗体重链和轻链可变区序列的寡核苷酸位点进行定向诱变。 以增加抗体结合亲和力或减小抗体免疫原性。
对于人源化抗体重链可变区（SEQ ID NO: 1)而言， 其中构架区 FR-H1内氨基酸 16的 A能被 E替换， 氨基酸 17的 S能被 T替换， 氨 基酸 20的 I能被 V替换； FR-H2内氨基酸 3的 K能被 R替换， 氨基 酸 9的 G能被 S替换； FR-H3内氨基酸 3的 T能被 V替换， 氨基酸 7 的 E能被 D替换， 氨基酸 10的 V能被 T替换， 氨基酸 14的 F能被 Y 替换， 氨基酸 19的 S能被 T替换， 氨基酸 27的 T能被 V替换。
对于抗体人源化轻链可变区（SEQ ID NO: 2)而言， 构架区 FR-L1内 氨基酸 11的 L能被 M替换，氨基酸 18的 R能被 E替换，氨基酸 21的 M 能被 I替换； FR-L2内氨基酸 13的 W能被 L替换； FR-L3内氨基酸 4的 S能被 A替换， 氨基酸 22的 L能被 V替换， 以及氨基酸 27的 A能被 F 替换。 通过导入上述氨基酸修饰中至少一个修饰， 设计多种人源化抗体 重轻链可变区的氨基酸序列， 部分人源化抗体重链可变区 VH、 轻链可 变区 VL氨基酸序列如表 1所示：
表 1 : 人源化抗体重链可变区 VH、 轻链可变区 VL氨基酸序列
Figure imgf000024_0001
实施例 5: pHu— anti-HlLl-TNFa人源化抗体表达载体的构建
1)人源化抗体轻链基因的构建 首先， 以人工合成方法制备人源化抗体轻链可变区 (F001VL) 的基 因片段， 制备的程序可先以轻链可变区的氨基 S史序列依其相对的遗传 密码子反转化取得核苷序列， 并于 5，终端加入 Kozak序列及轻链前导 序列， 再以人工合成方法制备成轻链可变区的基因片段。 将此基因片 段克隆入适当载体， 得 pHu-VLl质粒。 其后再以 pHu-VLl质粒作为模 板， 使用 5'引物 FVHX (SEQ ID NO: 17)和 3'引物 VKCKO (SEQ ID NO: 21)得到 5'片段， 含有人源化抗体的轻链可变区 (VL)和人 κ轻链恒定区 (CK) 5'端的 7个氨基酸的基因片段。 在此同时， 用从人白细胞制备的 RNA以合适的 5'引物 HuCKF (SEQ ID NO: 22)和 3'引物 HUCKB (SEQ ID NO: 23)通过逆转录和 PCR得到含人 K轻链恒定区（CK) 编码序列的 基因。 最后， 将含有人源化抗体的轻链可变区 (Vu)的片段与以上的人 CK基因通过 PCR,用 5'引物 (FVHX, SEQ ID NO: 17)和 3'引物 (HUCKB , SEQ ID NO: 23) , 将人源化的轻链可变区 (VU)和人 CK基因片段连接起 来， 得到含有轻链编码序列、 长度约为 700 bp的基因片段。 此基因片 段以内切酶 Hind III 和 Bam HI处理后， 插入载体如 PUC19(ref: Yanisch-Perron, C. , Vieira, J. and Messing, J. (1985) Gene, 33 , 103-119.), 用以表达人源化抗体的轻链蛋白。该基因片段的序列经 DNA测序验证 正确。
2) 人源化抗体重链基因的构建
首先， 以人工合成方法制备人源化抗体重链可变区 (E001VH)的基 因片段， 制备的程序可先以重链可变区的氨基 S史序列依其相对的遗传 密码子反转化取得核苷序列， 并于 5，终端加入 Kozak序列及重链前导 序列， 再以人工合成方法制备成重链可变区的基因片段。 将此基因片 段克隆入适当载体， 得 pHu-VHl质粒。 其后再以 pHu-VHl质粒作为模 板，使用 5'引物 FVHX (SEQ ID NO: 17)和 3'引物 RVCG (SEQ ID NO: 18) 得到 5'片段， 含有人源化抗体的重链可变区 (VH1)和人 IgGi重链恒定区 (Cyl) 5'端的 7个氨基酸的基因。 在此同时， 用从人白细胞制备的 RNA 以合适的 5'引物 HuCGF (SEQ ID NO: 19)和 3'引物 HUCGE (SEQ ID NO: 20)通过逆转录反应和 PCR得到含人 IgGi重链恒定区 编码序列 的基因。最后，将含有人源化抗体的重链可变区 (VH)的片段与以上的人 CYl基因通过 PCR,用 5'引物 (FVHX, SEQ ID NO: 17)和 3'引物 (HUCGE, SEQ ID NO: 20), 将人源化抗体的重链可变区 (VH1)和人 基因片段连 接起来， 得到含有重链编码序列、 长度约为 1400 bp的基因片段。 此基 因片段以内切酶 Hind III 和 EcoRl处理后， 插入载体如 PUC19(ref: Yanisch-Perron, C. , Vieira, J. and Messing, J. (1985) Gene, 33 , 103-119.), 用以表达人源化抗体的的重链蛋白。该基因片段的序列经 DNA测序验 证正确。
3)人源化抗体单一链表达载体
用上述方法中获得的重链和轻链的编码 cDNA 插入到 pcDNA3 (购 自 Invitrogen USA , Carlsbad , CA USA) 载体中 ， 构建 pHu_anti-HlLl-TNFa人源化表达载体。 该表达载体质粒含有在哺乳动 物细胞中高水平表达所需的巨细胞病毒早期基因启动因子-增强子。 同 时， 载体质粒中含有可选择标记基因， 从而在细菌中赋予氨苄青霉素 抗性， 而在哺乳动物细胞中赋予 G418 抗性。 另外， 载体质粒中含有 DHFR基因，在合适的宿主细胞中，能以氨曱喋呤 (Methotrexate, MTX, Sigma)共扩增嵌合抗体基因和 DHFR基因（参见， 例如 Axel, R.,等人 的美国专利 5,179,017 号； Kaufman,R.和 Sharp,P. , J.Mol. Biol. 159:601-621,1982)。 实施例 6: 人源化抗体的表达
将上述已构建的重组表达载体质粒转染入哺乳动物宿主细胞系， 以表达抗 hTNFa人源化抗体。 为了稳定高水平的表达， 优选的宿主细 胞系是二氢叶酸还原酶 (DHFR)缺陷型的中国仓鼠卵巢 (CHO)细胞 (参 见， 例如 Chasin,L.等人的美国专利 4,818,679号）。 优选的转染方法是 电穿孔， 也可以使用其他方法， 包括磷酸钙共沉降， 脂转染和原生质 融合等。 在电穿孔中， 用设为 250V电场和 960 μ¥ά电容的 Gene Pulser (Bio-Rad Laboratories),在比色杯内加入 2χ 107个细胞悬浮在 0.8 mL 的 PBS中， 并含有 l(^g用 PvuI(TakaRa)线性化的表达载体质粒 DNA。 转染 2 天后， 加入含有 0.2 mg/mL G418 以及 200 nM 氨曱喋呤 (methotrexate 或 MTX)。 为了实现较高水平的表达， 用受 MTX药物抑 制的 DHFR基因共扩增转染的人源化抗体基因。 用极限稀释亚克隆转 染子， 测定各细胞系的分泌率， 选出高水平表达人源化抗体的细胞株。 实施例 7: 抗体中和 TNFa对 L929细胞的杀伤作用研究
将 L929细胞用胰蛋白酶消化、 离心， 以 10%FCS的 1640培养基 重悬、计数，以一定浓度加至 96孔板 1〜11列，再加入合适浓度的 TNFa 于 96孔板 1〜10列， 分别以首孔浓度 0.4 ug/mL, 2倍梯度稀释阿达木 单抗 (购自雅培公司）、 氨基酸未修饰的嵌合抗体 AT(CE)-1、 以及按照 实施例 5和 6的方法制备的人源化抗体 AT132、AT135、AT143、AT151、 AT 164,每一抗体按从高到低 (1列至 9列）的浓度分别加至加至 96孔板 A、 B排， C、 D排， E、 F排和 G、 H排， 第 10列为 TNFa对照， 第 11列为细胞对照， 第 12列为培养基对照， 加好后置 37°C二氧化碳培 养箱中培养。 完成孵育后， 加入显色液， 孵育， 酶标仪检测吸光度， 结果见表 2所示。
表 2: 抗体中和 TNFa对 L929细胞的杀伤作用
Figure imgf000027_0001
表 2分析结果表明，进行 FR区突变后的人源化抗体仍然具有较好 的中和 TNFa的活性， AT 132的 EC50值与阿达木单抗相近， 为 20.4 ng/mL。 实施例 8: AT132中和 TNFa对人 U937细胞 (人淋巴瘤）的杀伤作 用研究
取状态良好的 U937细胞， 计数后以 10% FCS的 1640培养基调整 细胞浓度至 3.75xl04个 /孔， 按 75 μΐ/孔加入到 96孔培养板。 用含 120 ng/mL TNFa 的细胞培养液分别梯度稀释 AT132标准品和供试品， 使 其首孔浓度均为 600 ng/mL, 稀译梯度 1.5倍， 稀译完毕后分别以 25 μΐ/孔加至 96孔板， 置 37°C二氧化碳培养箱中培养 40小时。 孵育完成 后， 每孔加入 10 μ1 0 Κ8显色液， 孵育 3h, 酶标仪 490nm/630nm双 波长检测， 以四参数进行曲线拟合， 统计标准品和供试品的 ED50 , 计算比活性 (公式： 100%x标准品 ED50/供试品 ED50)。
图 1为抗体中和 TNFa杀伤 U937细胞作用曲线
图 1分析结果表明， 在 AT132极低浓度的情况下 TNFa对细胞基 本处于杀伤状态， 随着 AT132 浓度的增高， TNFa 的作用逐渐被拮 抗， 至 AT 132浓度约 80 ng/mL时 TNFa的杀细胞作用基本被完全拮 抗， 因此存在明显的剂量依赖性， 根据多次试验的结果， 其中和 30 ng/mL TNFa的半数有效浓度平均为 24.1 ng/mL。 实施例 9: AT132亲和力测定
应用 Biacore X 100测定， Biacore X 100动力学 /亲和力软件分析， 采用间接捕获的方法， 通过 Amine Coupling Kit把羊抗人 IgG Fc多克 隆抗体偶联在 CM5 芯片表面作为捕获分子， 通过计算分别把 AT132 和对照阿达木单抗稀译到一定浓度作为配体， 把 TNFa作为分析物。 分析物稀译 5个浓度， 每个浓度作为一个循环， 先用 HBS-EP緩冲液 运行 3个循环， 设计一个分析物浓度为 0浓度运行 2个循环， 最后设 计一个重复的分析物浓度运行 1个循环。 整个过程运行 11个循环， 每 个循环都可绘制一条曲线， 通过 Biacore X100动力学 /亲和力分析软件 分析得到所测抗体阿达木单抗的动力学 /亲和力数据。
测定结果： AT132 解离常数 (Kd)为 1.19x lO_uM, 即亲和常数 (Ka) 为
Figure imgf000028_0001
, 阿达木单抗解离常数为 1.08x l0-1QM, 即亲和常数为 9.3χ 109Μ 。 实施例 10: AT132与鼠 TNFa和猴 TNFa结合活性
分另' J包被重组人 TNFa 5 ng/孔， 小鼠 TNFa 25 ng/孔， 猴 TNFa 5 ng/孔， 室温封闭 l h, 洗涤， 然后加入 1.8倍梯度稀释的 AT 132, 起始 浓度为 250 ng/mL, 25 °C孵育 2 h, 洗涤， 力口入 HRP标记的抗人 Fc抗 体， 室温孵育 1 h, 洗涤后加入底物溶液， 100 μΐ/well, 37°C避光 30 min, 按照加显色液的顺序， 加入 0.2 M H2S04, 50 μΐ/well, 终止反 应。 终止约 5分钟内， 在酶标仪上 450 nm/630 nm测 OD值， 采用四参 数拟合， 得出半数有效浓度， 比较 AT132与不同种属来源的 TNFa的 结合活性。
与小鼠 TNFa结合活性的比较研究结果表明， AT132与小鼠 TNFa 无结合， 根据包被浓度和 AT132加样浓度计算， 其与小鼠 TNFa结合 活性至少比与人 TNFa的结合活性低 1000倍。
与猴 TNFa结合活性的比较研究结果表明， 结果与猴 TNFa结合活 性约为与人 TNFa结合活性的 50%。 实施例 11: 注射剂的制备
AT132、 AT135注射剂制备方法如下：
1) 制备 20 L緩冲液 (相当于 20.180 kg-溶液的密度： 1.009 g/mL) 称量出以下重量的成分： 240.0 g甘露糖醇、 26.1 g—水合柠檬酸、
6.1 g 4宁檬酸钠、 30.6 g二水合磷酸氢二钠、 17.2 g二水合磷酸二氢钠、 123.3 g 氯化钠、 20.0 g 失水山梨醇聚氧乙烯（20)醚油酸酯和 19715.7-19716.1 g的水。
通过将 40.0 g的氢氧化钠与 1000.8 g的注射用水混合制备氢氧化 ]溶液。
然后，通过将以下预先称量好的成分 (如上文所述)溶解在大约 90% 的注射用水中制备緩冲液： 甘露糖醇、 一水合柠檬酸、 柠檬酸钠、 二 水合磷酸氢二钠、磷酸二氢钠、 氯化钠和失水山梨醇聚氧乙烯 (20)醚油 酸酯。
在添加上述所有緩冲液成分之后， 用按上述方法制备的 1 M氢氧 化钠调节该溶液的 pH。 在添加氢氧化钠之后， 添加最终重量的水。 然 后通过过滤膜 (亲水性聚偏二氟乙烯， 0.22 μιη孔径)将緩冲液过滤到无 菌容器中。 所使用的过滤介质是过滤消毒的氨气。
2) 制备 40 L制剂 (相当于 40.88 kg)
将过滤的緩冲溶液添加到按以下方法制备的解冻的并且合并的抗 体浓缩物 (所述药物制剂的活性成分)中。在制备所述药物制剂之前的水 浴中对所述抗体 (浓缩物)进行解冻。 使用 34.207 g抗体浓缩物， 它相当 于 2.0 kg的蛋白， 浓度为 60 mg蛋白 /mL蛋白浓缩物。 所述浓缩物的 密度为 1.0262 g/mL。 可以使用 25.655-37.316范围内的任何蛋白浓缩 物， 它相当于蛋白浓缩物中蛋白的浓度为 50-80 mg/mL。在搅拌状态下 添加所述緩冲液， 直到达到总溶液的最终重量。
然后按照上述方法， 过滤包含它的所有成分的制剂， 所不同的是 所述制剂是通过两层无菌 0.22 μιη的膜过滤器过滤的。在消毒之后，对 所述制剂进行包装， 以便在管形瓶或预先填充的注射器中使用。 实施例 12: AT132小鼠急性毒性试验
受试样品： AT132冻干粉针，规格： 20 mg/瓶；辅料对照品： AT132 緩冲液 (含组氨酸、 海藻糖)； 溶剂： 灭菌注射用水。 试验动物分组和给 药剂量： 4-6周龄昆明 (KM)小鼠 60只， 18-22g, 雌雄各半， SPF级。 将以上动物随机分为 3组， 每组 20只， 雌雄各半。 单次给药， 观察 14 天。 按表 3所示给药剂量和给药途径：
表 3: 给药剂量和给药途径 给药剂量 给药容量
组号 组别
(mg/kg) (mL/kg)
1 辅料对照组 一 一
2 供试品皮下注射组 500 25
3 供试品静脉注射组 500 25 观察指标： 体重、 食量、 精神状态、 行为活动、 粪便性状等， 试 验观察期结束后， 将动物实施安乐死并进行病理学大体解剖观察， 发 现异常组织或器官进行组织学检查。
结果： 试验期间， 所有动物未发生死亡或濒死； 所有动物精神状 态良好， 行为活动、 饮食、 饮水正常， 未见明显异常表现。 各给药组 动物的体重未见有与给药相关的规律性改变。 病理学大体解剖观察未 见与给药相关的异常变化。
结论： 在本试验条件下， AT132粉针以 500 mg/kg的剂量单次静脉 和皮下注射给予小鼠， 未见明显毒性反应， 最大耐受剂量 (MTD)大于 500 mg/kg。 实施例 13: AT132对 D-氨基半乳糖致敏小鼠 rhTNFa诱导致死保 护作用 取 C57BL/6小鼠 51只， 体重为 20.0士 2.0 g, 分为 6组 (见表 4)。 第 2-5组小鼠每只分别腹膜内注射 ATI 32溶液 0.25 mL, 其中， 第 2组小 鼠每只给药剂量以 AT132计是 5.2 g/只， 第 3组小鼠每只给药剂量以 ATI 32计是 26 g/只， 第 4组小鼠每只给药剂量以 AT 132计是 52 μ^/ 只， 第 5组小鼠每只给药剂量以 AT132计是 26 g/只； 第 1组小鼠每 只腹膜内均注射 pH 5.63的柠檬酸盐緩冲液 0.25 mL,第 6组小鼠每只 腹膜内均注射 0.25 mL人 IgGi HuIgGi , 阴性对照）， 以 HulgGi计是 26 g/只。 30 分钟后各组小鼠 (除第 5 组外)每只均腹膜内注射 0.25 mL rhTNFa (Primegene, 批号 1030109021)和 D-氨基半乳糖混合物溶液， 第 5组小鼠腹膜内注射 0.25 mL緩冲液， 观察 48小时的小鼠致死数， 计算存活率如表 4所示。
表 4: 各组小鼠存活率
Figure imgf000031_0001
结果： 緩冲液和 HulgGi组小鼠存活率为 0, 而 AT132剂量依赖地 保护了 rhTNFa和 D-氨基半乳糖致敏小鼠。 因此， ATI 32对 D-氨基半 乳糖致敏小鼠 rhTNFa诱导致死有保护作用。 实施例 14: AT132对大鼠 II型胶原诱导的关节炎的作用研究 取 Wistar雌性大鼠 50只随机分为 4组， 每组均为 10只。 分别为 空白对照组、 致炎对照组 (即模型组）、 ATI 32 l mg/kg组、 AT 132 5 mg/kg组。
除空白对照组， 其余各组分别在大鼠背部皮内注射 II型胶原免疫， 致炎对照组注射 0.1 mol/L醋酸及完全或不完全弗氏佐剂乳化液 (Sigma 公司， 4比号 129K8701). AT132 1 mg/kg, AT 132 5 mg/kg组于注射免疫当天开始腹腔注射给 药。 于第 1次免疫后一周再以同样方式加强免疫一次， 共给药 28天。 给药前及给药后不同时间用 YLS-7B足趾容积测量仪测量大鼠足关节 肿胀值。 实验结束后取足关节做病理检查。 然后计算肿胀率及抑制率， 组间差异以 t检验进行比较。 计算公式如下： 肿胀率％ = E"—E。 X 100%
E
En=致炎后不同时间的肿月长值
E0=致炎前的肿长值
实验数据以均数和标准差 (s)表示，采用 t检验进行分析统计。结果 见图 2所示。 AT132 1 mg/kg组显示明显的抑制效果， 在第 19天开始 即有较好疗效， 且此时疗效最佳为 63.10%, 此后抑制效果逐渐降低， 在第 28天仍有比较好的效果， 抑制率为 55.71%; AT 132 5 mg/kg组显 示出明显的抑制效果， 从第 19天开始有显著疗效， 大鼠足关节红肿逐 渐消退， 活动趋于正常， 疗效一直持续到第 28天，抑制率第 21d最高， 为 47.27%, 其它均为 40%左右。 实施例 15: AT132对 Tg197小鼠关节炎模型的药效学研究 本实验选用 Tgl97转基因小鼠 (购自 Cyagen Biosciences), 将小鼠 分为 6组， 每组 10只小鼠， 雌雄各半。 具体为： 组 1 : AT 132 1 mg/kg; 组 2: 溶剂组 (含柠檬酸和氯化钠的緩冲液)； 组 3: AT132 30 mg/kg; 组 4: AT132 10 mg/kg); 组 5: 阿达木单抗 10 mg/kg; 组 6: ATI 32 3 mg/kg。 另外选择 4只 Tgl97小鼠做为空白对照组。
给 3周龄 Tgl97小鼠腹腔内注射 AT132, 每周 2次， 至 10周龄； 给药前用緩冲液将 AT132稀释至所需的浓度， 每组给予受试药物的剂 量为 ΙΟ μΙ/g体重， 观察小鼠关节炎程度， 对小鼠棵关节进行病理学评 分， 艮据关节炎评分和病理评分计算抑制率。
1) AT132对 Tgl97小鼠关节炎程度的研究
每周对小鼠关节形态变化进行评分以评估关节炎程度， 具体关节 炎评分标准如下：
0.0 = 没有关节炎 （外观正常， 小鼠能够支持身体的体重， 整 体的灵活性 /逃避能力正常， 握力最大） ；
0.5 =发病的关节炎（关节爪子轻微肿胀， 外观正常， 小鼠能够 支持身体的体重， 整体的灵活性 /逃避能力正常， 握力最大） ；
1.0 =轻度关节炎 （关节肿胀变形， 爪子红肿， 外观正常， 小鼠 能够支持身体的体重， 整体的灵活性 /逃避能力正常， 握力最大）；
1.5 =轻度到中度关节炎 （ 关节爪子肿胀， 变形 + 最后手指向 内变形， 简短支持上部的重量边缘是 /否， 整体灵活性减低， 握力减 少 ） ；
2.0 = 中度关节炎 （严重关节、 爪子和手指肿胀， 脚关节变形 ， 不能支持上部体重跌倒， 整体灵活性消失， 握力消失， 爬行 /进食受影 响 ） ；
2.5 = 中度到严重关节炎 （严重关节、 爪子和手指肿胀， 脚关节 变形， 不能支持上部体重跌倒， 整体灵活性消失， 握力消失， 爬行 / 进食受影响 +手指爪子变形， 小鼠活动受损 ） ；
3.0 =严重关节炎 （关节僵硬， 检测到弯曲和严重活动受损， 小 鼠垂死挣扎)。
Tgl97小鼠关节炎在体评分如图 3A所示：
2) Tgl97小鼠的踝关节组织病理学研究
为了监测疾病的状态， 本试验的 4只同窝出生的 Tgl97小鼠 (编号 为 Conl-Con4)在 3周龄时处死， 作为治疗开始的关节样本。 实验小鼠 于 10周龄时处死，取踝关节切片， 苏木 /伊红染色好后以盲法的方式进 行关节炎的显微镜组织病理得分评价， 其评级为 0-4, 如下：
0 =无可检测的病理
1 =滑膜增生， 多形核白细胞存在浸润
2 =血管翳及纤维组织的形成和病灶软骨下骨质侵蚀
3 =软骨破坏和骨质侵蚀
4 =扩大的软骨破坏和骨质侵蚀
Tgl97小鼠的组织病理学分析如图 3B、 3C所示
3)根据关节炎和病理评分计算抑制率。 关节炎评分抑制率％ = 0 " X 100%
E,
En=各组关节炎评分
E0=溶剂组关节炎评分
根据病理学评分计算抑制率， 计算公式如下: 病理学评分抑制率％ = Eo—E" X 100%
En=各组病理学评分
E。=溶剂组病理学评分
结果见表 5所示。
表 5: 小鼠关节炎评分抑制率和病理评分抑制率研究
Figure imgf000034_0001
结果： 给予不同剂量 AT132(组 1 , 组 3 , 组 4, 组 6)后， 对类风湿 关节炎表现出显著的抑制作用， 呈现出典型的量效作用。 特别是从组 1(1 mg/kg)到组 6(3 mg/kg), 其抑制作用能够明显的区别开来。从组 6(3 mg/kg)到组 4(10 mg/kg), 其抑制作用又明显的区别开来。 只有组 4(10 mg/kg)与最高剂量组 3(30 mg/kg)间的抑制作用没有区别。 重要的是在 10 mg/kg相同剂量水平，AT132 治疗组 4 与阿达木治疗组 5间的抑制 作用没有显著性差异， 与溶剂组 2相比对关节炎的组织病理有明显改 善作用。 实施例 16： AT132单克隆抗体组织交叉反应
1) AT132单克隆抗体人体组织交叉反应
将人鼻息肉石蜡切片及四套正常人体组织 (供体 A、 B、 C、 D, 由 中国食品药品检定研究院， 国家药物安全评价监测中心提供)石蜡切片 分为 3组， 分别为实验组 (生物素标记 AT132 )、 阳性对照组 (生物素标 记阿达木单抗）、 阴性对照组 (緩冲液 PBS), 观察组织交叉反应后的组 织切片的着色情况。
实验结果表明， 阴性对照组人鼻息肉以及正常人体各组织均无染 色。 生物素-阿达木单抗阳性对照组与人体鼻息肉的巨噬细胞、 正常人 体淋巴结的巨噬细胞以及肺脏的肺泡巨噬细胞可见弱到中度染色； 其 余组织无染色。 生物素 -AT132实验组与人体鼻息肉的巨噬细胞可见弱 到中度染色； 正常人体组织交叉反应与阿达木单抗具有相似结果。
2) AT132单克隆抗体食蟹猴组织交叉反应
将人鼻息肉石蜡切片及三套正常食蟹猴组织 (供体 A、 B、 C, 由 中国食品药品检定研究院， 国家药物安全评价监测中心提供)石蜡切片 分为 3组， 分别为实验组 (生物素标记 AT132 )、 阳性对照组 (生物素标 记阿达木单抗）、 阴性对照组 (緩冲液 PBS), 观察组织交叉反应后的组 织切片的着色情况。
实验结果表明， 阴性对照组人鼻息肉以及正常食蟹猴各组织均无 染色。 生物素 -阿达木单抗阳性对照组与人体鼻息肉的巨噬细胞可见弱 到中度染色； 正常食蟹猴组织无染色。 生物素 -AT132实验组与人鼻息 肉的巨噬细胞可见弱到中度染色。 正常食蟹猴组织交叉反应与阿达木 单抗具有相似结果。
Figure imgf000036_0001
下面的说明与本申请说明书中此处提到的
保藏的微生物或其他生物材料相关：
-1 页码 11
-2 行号： 28
-3 保藏事项
-3-1 保藏单位名称 CCTCC 中国典型培养物保藏中心
-3-2 保藏单位地址 中国湖北省武汉市武汉大学， 邮政编码: 430072， Hubei
(GN)。
-3-3 保藏日期 2011年 3月 07日 （07.03.2011)
-3-4 保藏号 CCTCC C201117
-5 本说明是对下列指定国
所有指定国
由受理局填写
-4 本表格与国际申请一起收到：
(是或否）
-4-1 受权官员 由国际局填写 -5 国际局收到本表格日期： -5-1 受权官员

Claims

权 利 要 求 书
1. 一种抗肿瘤坏死因子人源化单克隆抗体， 其中所述抗体包含重 链和轻链， 所述重链具有 SEQ ID NO: 1所示或与其具有至少 75%的同 源性的氨基酸序列， 且所述轻链具有 SEQ ID NO: 2所示或与其具有至 少 75%的同源性的氨基酸序列。
2. 一种抗肿瘤坏死因子人源化单克隆抗体， 其中所述抗体包含重 链和轻链， 所述重链具有 SEQ ID NO: 1所示或与其具有至少 75%的同 源性的氨基酸序列， 且所述轻链具有 SEQ ID NO: 2所示或与其具有至 少 75%的同源性的氨基酸序列， 其中所述重链和 /或轻链通过插入、 缺 失或保守性置换其中的 1 ~ 5个氨基酸残基进行修饰。
3. 如权利要求 1或 2所述的抗肿瘤坏死因子人源化单克隆抗体， 其 中所述重链可变区的互补决定区 CDR-H1为 SEQ ID NO: 3所示的氨基 酸序列； CDR-H2为 SEQ ID NO: 4所示的氨基酸序列； CDR-H3为 SEQ ID NO: 5所示的氨基酸序歹l ;其中构架区 FR-H1内氨基酸 16的 A能被 E 替换，氨基酸 17的 S能被 T替换，氨基酸 20的 I能被 V替换 (SEQ ID NO: 11)； FR-H2内氨基酸 3的 K能被 R替换，氨基酸 9的 G能被 S替换 (SEQ ID NO: 12); FR-H3内氨基酸 3的 T能被 V替换， 氨基酸 7的 Ε能被 D 替换， 氨基酸 10的 V能被 Τ替换， 氨基酸 14的 F能被 Υ替换， 氨基酸 19的 S能被 Τ替换， 氨基酸 27的 Τ能被 V替换 (SEQ ID NO: 13); 以及 该人源化抗体的轻链可变区的互补决定区 CDR-L1为 SEQ ID NO: 6所 示氨基酸序列； CDR-L2为 SEQ ID NO: 7所示氨基酸序列； CDR-L3为 SEQ ID NO: 8所示氨基酸序列， 其中构架区 FR-L1内氨基酸 11的 L能 被 M替换，氨基酸 18的 R能被 E替换，氨基酸 21的 M能被 I替换 (SEQ ID NO: 14); FR-L2内氨基酸13的W能被L替换(SEQ ID NO: 15); FR-L3 内氨基酸 4的 S能被 A替换， 氨基酸 22的 L能被 V替换， 以及氨基酸 27的 A能被 F替换 (SEQ ID NO: 16)。
4. 编码如权利要求 1-3中任一项所述的抗肿瘤坏死因子人源化单 克隆抗体的核酸序列。
5. 包括如权利要求 4所述的核酸序列的载体。
6. 如权利要求 5所述的载体，其中所述载体也包括与所述核酸可操 作性连接以有利于其表达的启动子。
7. 包含如权利要求 5或 6所述的载体的宿主细胞。
8. 如权利要求 1-3中任一项所述的抗肿瘤坏死因子人源化单克隆 抗体在制备用于 hTNFa的诊断性分析的药物中的应用。
9. 如权利要求 1-3中任一项所述的抗肿瘤坏死因子人源化单克隆 抗体在制备用于治疗 hTNFa有关的疾病的药物中的应用。
10. 如权利要求 9所述的应用，其中所述的 hTNFa有关的疾病是脓 毒症、 自身免疫性疾病、 恶性肿瘤、 肺功能紊乱、 移植排斥、 细菌性 脑膜炎、 脑型疟、 AIDS和 AIDS相关综合症 (ARC；)、 移植继发的巨细 胞病毒感染。
11. 包括如权利要求 1 -3中任一项所述的抗肿瘤坏死因子人源化单 克隆抗体的药物组合物， 其中所述药物组合物包括抗 hTNFa人源化抗 体和可药用赋形剂。
PCT/CN2012/071079 2011-02-28 2012-02-13 抗肿瘤坏死因子α的人源化抗体 WO2012116595A1 (zh)

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BR112013021860-6A BR112013021860A2 (pt) 2011-02-28 2012-02-13 anticorpo anti-tnfalpha humanizado
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CA2827799A CA2827799C (en) 2011-02-28 2012-02-13 Tumor necrosis factor-a humanized antibody
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JP6532136B2 (ja) * 2014-06-09 2019-06-19 株式会社カネカ 抗体に結合する低分子化合物のスクリーニング方法
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