WO2020224529A1 - 低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用 - Google Patents

低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用 Download PDF

Info

Publication number
WO2020224529A1
WO2020224529A1 PCT/CN2020/088253 CN2020088253W WO2020224529A1 WO 2020224529 A1 WO2020224529 A1 WO 2020224529A1 CN 2020088253 W CN2020088253 W CN 2020088253W WO 2020224529 A1 WO2020224529 A1 WO 2020224529A1
Authority
WO
WIPO (PCT)
Prior art keywords
monoclonal antibody
tcx060
tnf
antibody
amino acid
Prior art date
Application number
PCT/CN2020/088253
Other languages
English (en)
French (fr)
Inventor
孙乐
任文林
Original Assignee
北京天成新脉生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京天成新脉生物技术有限公司 filed Critical 北京天成新脉生物技术有限公司
Priority to AU2020270242A priority Critical patent/AU2020270242B2/en
Priority to US17/609,778 priority patent/US20230242634A1/en
Priority to EP20801746.7A priority patent/EP3967708A4/en
Priority to JP2021566946A priority patent/JP7300008B2/ja
Publication of WO2020224529A1 publication Critical patent/WO2020224529A1/zh

Links

Images

Classifications

    • 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
    • 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
    • A61P37/04—Immunostimulants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of biotechnology, in particular to the anti-TNF- ⁇ humanized monoclonal antibody TCX060 with low immunogenicity and low ADCC/CDC function and its application.
  • Tumor necrosis factor alpha is a cytokine secreted by immune cells and naturally secreted in inflammation and immune response.
  • TNF- ⁇ has a tendency to up-regulate in patients with various chronic diseases (such as Crohn’s disease, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, etc.) (for example, in rheumatoid In the synovial fluid of patients with arthritis (RA), the level of TNF- ⁇ is elevated), and it plays an important role in the process of pathological inflammation and joint destruction.
  • Human TNF- ⁇ has a molecular weight of 17kDa and can exist in the body as a monomer or trimer, and its active form is a trimer.
  • TNF- ⁇ exerts biological activity by interacting with cell surface receptors p55 and p75.
  • monoclonal IgG antibodies or soluble TNF- ⁇ receptors are generally used to neutralize TNF- ⁇ in the body.
  • TNF- ⁇ mainly include etanercept, inflix, adalim, and cytrol, among which etanercept is produced by two human TNF- ⁇ receptors.
  • Inflixi can bind to monomeric (inactive) and trimer (active) types of soluble TNF- ⁇ ; Etanercept is more inclined It binds to active polymeric forms of TNF- ⁇ and TNF- ⁇ , while the Adalim monoclonal antibody only binds to TNF- ⁇ , not TNF- ⁇ .
  • Certolizumab developed by Celltech UCB, is a humanized monoclonal antibody that specifically binds to TNF- ⁇ and blocks its interaction with cell surface TNF receptors p55 and p75.
  • the Certolizumab monoclonal antibody has strong immunogenicity in the host body, and up to 23% of patients using this monoclonal antibody will have an immune response. Immune response may cause immune complex-mediated antibodies or fragments to be eliminated from the circulation, and cause repeated administration to be unsuitable for treatment, thereby reducing the patient's therapeutic benefit and limiting the re-administration of antibodies.
  • the original research Cytozhu needs to be used in conjunction with immunosuppressive agents, which greatly increases the risk of side effects.
  • the purpose of the present invention is to provide an anti-TNF- ⁇ humanized monoclonal antibody with low polymer, low immunogenicity and low ADCC/CDC function, its preparation method and its application .
  • the present invention analyzes the amino acid sequence of the Cytozine monoclonal antibody by using commercial DNAStarTM software.
  • the immunogenicity of the sequence is very low, but the PEGylated Cytozine monoclonal antibody may lead to the production of multimers, which in turn leads to increased immunogenicity, that is, although PEGylation reduces ADCC/CDC function, it leads to immunity The question of originality.
  • the present invention transforms the monoclonal antibody sequence on the basis of the Cytozine monoclonal antibody sequence, so as to maintain the high TNF- ⁇ binding affinity and specificity of the Cytozhu, while reducing its content.
  • Polymer production and immunogenicity as well as ADCC/CDC functions, specific modifications include:
  • the Cytozine monoclonal antibody was transformed into a human IgG1 full-length antibody.
  • protein structure analysis software such as Pymol to analyze the structure of the antibody, look for a relatively flexible region between the variable region and the constant region of the antibody to carry out flexible amino acid insertion and transformation; finally through the heavy chain CDR3 region and the antibody A flexible amino acid fragment is inserted between the CH2 region to cut off the mechanical stress transmission generated by the antibody variable region after binding to the antigen, so that the heavy chain constant region of the antibody cannot be fully exposed to the Fc receptor and/or complement binding site, thereby weakening the antibody It binds to killer cells or complement expressing IgG Fc receptors such as NK cells, macrophages and neutrophils, so that the antibody cannot induce or reduce the signals that induce ADCC and CDC.
  • IgG Fc receptors such as NK cells, macrophages and neutrophils
  • the first objective of the present invention is to provide an anti-TNF- ⁇ humanized monoclonal antibody TCX060, which is obtained by modifying the Certolizumab monoclonal antibody as follows:
  • the full-length light chain sequence and the heavy chain variable region sequence of the Certolizumab monoclonal antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively;
  • the anti-TNF- ⁇ humanized monoclonal antibody TCX060 has the function of binding to human TNF- ⁇ and blocking the binding of human TNF- ⁇ to TNF receptors.
  • step (3) inserting flexible amino acid fragments between the CDR3 and CH2 regions of the obtained human IgG1 subtype full-length antibody can block the stress transmission between the variable region and the constant region of the antibody, so that the Fc of the antibody is affected.
  • the body and/or complement binding site cannot be fully exposed, thereby effectively reducing the ADCC/CDC effect of the antibody.
  • the insertion position of the flexible amino acid fragment is the 237th and 238th positions of the sequence shown in SEQ ID NO.3 Between amino acids.
  • the flexible amino acid fragment contains one or more glycine or serine.
  • sequence of the flexible amino acid fragment is GGGS, GGSGGS or GSGSGS.
  • the insertion position of the flexible amino acid fragment is between the 237th and 238th amino acids of the sequence shown in SEQ ID NO. 3; the sequence of the flexible amino acid fragment is GGGS.
  • the present invention performs a large number of screenings for the heavy chain of the anti-TNF- ⁇ humanized monoclonal antibody obtained based on the above modification, and obtains the anti-TNF- ⁇ humanized monoclonal antibody with the best performance in terms of immunogenicity and ADCC/CDC function.
  • amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids means that one or more amino acid residues are different from the sequence shown but retained.
  • the biologically active sequence of the molecule which can be a "conservatively modified variant” of the full length of the heavy chain with the amino acid sequence shown in SEQ ID NO. 4 or modified by "conservative amino acid substitution", " Conservatively modified variants” or “conservative amino acid substitutions” refer to amino acid substitutions known to those skilled in the art, and making such substitutions generally does not change the biological activity of the resulting molecule.
  • substitutions are preferably performed in accordance with the substitutions shown in Table 1:
  • the heavy chain of the above sequence shown in SEQ ID NO. 4 can be combined with the light chain of the modified or unmodified Certolizumab monoclonal antibody, effectively reducing the ADCC/CDC function of the non-PEGylated Certolizumab monoclonal antibody, and has Low immunogenicity.
  • the present invention obtains an optimal human TNF- ⁇ monoclonal antibody TCX060 composed of the full-length light chain and heavy chain.
  • the long amino acid sequence is shown in SEQ ID NO. 4, and the full-length light chain amino acid sequence is shown in SEQ ID NO. 1.
  • This monoclonal antibody retains the antigen binding site of the Certolizumab antibody and has a similar expression level to the Certolizumab antibody.
  • the second object of the present invention is to provide a gene encoding the anti-TNF- ⁇ humanized monoclonal antibody TCX060.
  • the gene encoding the anti-TNF- ⁇ humanized monoclonal antibody TCX060 includes any nucleic acid encoding the heavy or light chain of the anti-TNF- ⁇ humanized monoclonal antibody provided by the present invention; according to the degeneracy of the codons, it can be The nucleic acid encoding the full-length amino acid sequence of the light chain and the heavy chain is obtained by codon optimization according to any host codon preference.
  • nucleotide sequence encoding the full length of the heavy chain is shown in SEQ ID NO.5
  • nucleotide sequence encoding the full length of the light chain is shown in SEQ ID NO.6.
  • the third object of the present invention is to provide a biological material containing the gene encoding the anti-TNF- ⁇ humanized monoclonal antibody TCX060.
  • the biological materials include expression cassettes, vectors, host cells, engineered bacteria or cell lines.
  • the fourth object of the present invention is to provide a preparation method of the anti-TNF- ⁇ humanized monoclonal antibody TCX060, which is achieved by expressing the genes encoding the full length of the heavy chain and the full length of the light chain.
  • the preparation method of the anti-TNF- ⁇ humanized monoclonal antibody TCX060 includes the following steps:
  • the fifth object of the present invention is to provide the anti-TNF- ⁇ humanized monoclonal antibody TCX060 or the anti-TNF- ⁇ humanized monoclonal antibody TCX060 gene or the biological material containing the gene is prepared for human Application of TNF- ⁇ as the target medicine.
  • the drug targeting human TNF- ⁇ is a drug for preventing or treating tumors, inflammations or autoimmune diseases.
  • the diseases include but are not limited to Crohn's disease, rheumatoid arthritis, psoriatic arthritis, active ankylosing spondylitis and the like.
  • the sixth object of the present invention is to provide the anti-TNF- ⁇ humanized monoclonal antibody TCX060 or the anti-TNF- ⁇ humanized monoclonal antibody TCX060 gene or the biological material containing the gene for preparing human TNF -Application of ⁇ detection reagents.
  • the seventh object of the present invention is to provide a drug or detection reagent containing the anti-TNF- ⁇ humanized monoclonal antibody TCX060.
  • the drug containing the anti-TNF- ⁇ humanized monoclonal antibody TCX060 may also contain other active ingredients or excipients permitted in the pharmaceutical field.
  • the present invention obtains the anti-TNF- ⁇ humanized monoclonal antibody through artificial modification on the basis of the Cytozil monoclonal antibody.
  • the humanized anti-TNF- ⁇ monoclonal antibody provided by the present invention has the same characteristics as the Cytozil monoclonal antibody The TNF- ⁇ antigen binding site, but its antibody conformation and immunogenicity are different from Cytozhu.
  • the humanized anti-TNF- ⁇ monoclonal antibody TCX060 provided by the present invention retains the antigen affinity and specificity of the Cytozhu monoclonal antibody, and its ADCC/CDC induction function is similar to that of the PEGylated Cytozhu monoclonal antibody, but The immunogenicity is significantly lower than that of the Cytozine monoclonal antibody; at the same time, the production process of the humanized anti-TNF- ⁇ monoclonal antibody provided by the present invention is more simplified. Lower immunogenicity will reduce the risk of drug side effects caused by the immune response caused by antibody immunogenicity in the human body. At the same time, the reduction of immunogenicity will prolong the humanized anti-TNF- ⁇ monoclonal antibody provided by the present invention.
  • the in vivo half-life of the prepared antibody drug avoids the reduction of drug efficacy caused by the antibody drug ADA to a greater extent; on the one hand, it can reduce the dosage of antibody drug and reduce the treatment cost; on the other hand, it can get rid of anti-TNF- ⁇ monoclonal antibody drugs and
  • the combined administration mode of immunosuppressive agents realizes the effect of single administration of anti-TNF- ⁇ monoclonal antibody drugs, removes the side effects caused by the combined use of immunosuppressive agents, and improves the safety of medication.
  • the humanized anti-TNF- ⁇ monoclonal antibody TCX060 provided by the present invention has great application potential and value, and is expected to become an ideal biological targeted therapeutic antibody.
  • Figure 1 is a diagram showing the results of double enzyme digestion and electrophoresis of the expression vector encoding the gene encoding the monoclonal antibody of Cytozil in Example 2 and the H2L0 monoclonal antibody encoding gene provided by the present invention, where a is the expression vector of the monoclonal antibody of Cytozil Light chain L0 plasmid Hind III and EcoRI double enzyme digestion results, the lanes from left to right are: plasmid before digestion, plasmid after digestion and DNA marker; b is the heavy chain H2 plasmid Hind of monoclonal antibody TCX060 provided by the present invention III and EcoRI double digestion results, the lanes from left to right are: plasmid before digestion, plasmid after digestion, and DNA marker.
  • Figure 2 is an SDS-PAGE electrophoresis diagram of the monoclonal antibody TCX060 in Example 4 of the present invention.
  • the lanes from left to right are: expression supernatant, Marker, and purified antibody.
  • FIG. 3 monoclonal antibody affinity TCX060 EC embodiment 50 of the present invention; H2L0 wherein the representative TCX060.
  • FIG. 4 shows the results of the TNF- ⁇ -mediated cell killing experiment of monoclonal antibody TCX060 in Example 5 of the present invention
  • Adalimumab represents the original research drug of Adalimumab
  • Certolizumab represents the original research drug of Certozumab
  • H2L0 represents the antibody TCX060H2L0 of the present invention.
  • Figure 5 is the mouse ADA evaluation result of the monoclonal antibody TCX060 in Example 6 of the present invention; among them, Certolizumab Day 0, Certolizumab Day 7, Certolizumab Day 14 represent the original Cytoliz original drug before immunization, and the seventh and 14th after immunization.
  • Day serum samples, H2L0Day0, H2L0Day 7, H2L0Day 14 respectively represent serum samples before immunization with the antibody drug TCX060H2L0 of the present invention, 7th and 14th days after immunization.
  • the present invention uses commercial DNAStarTM software to analyze and evaluate the original sequence of the Cytoball monoclonal antibody, and the analysis result shows that the immunogenicity of the original Cytoball monoclonal antibody sequence is very low. It is inferred from this that the PEGylation of the original Cytobes monoclonal antibody to remove ADCC/CDC function increases the multimer of the antibody, which in turn leads to the ADA of the drug as high as 23%. Therefore, it is necessary to remove the PEGylation modification of the original Cytozub monoclonal antibody while reducing its ADCC/CDC function.
  • the amino acid sequences of the full length of the light chain and the variable region of the heavy chain of the original Cytobes are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • the nucleotides encoding the heavy chain H2 and light chain L0 were designed Sequence (where the nucleotide sequence of L0 is shown in SEQ ID NO. 6, and the nucleotide sequence of H2 is shown in SEQ ID NO. 5), the restriction sites on both sides of the light chain sequence are designed as Hind III+EcoR I.
  • connection vector is pUC57.
  • pEE12.4 for heavy chain expression
  • pEE6.4 for light chain expression
  • the above expression vector and synthesized gene sequence were respectively subjected to corresponding double enzyme digestion, and the target gene obtained by digestion And the expression vector were separated by agarose gel electrophoresis, and then the target band was cut out and recovered with Qiagen Gel Extraction Kit, ligated according to T4 DNA enzyme ligation system at 16°C overnight, and then transformed into E. coli DH5 ⁇ , and the obtained transformants were identified by PCR With plasmid extraction and sequence determination, expression vectors carrying full-length heavy chain H2 and light chain L0 genes were obtained.
  • the pEE12.4 expression vector carrying the original Cytobead heavy chain H0 (sequence shown in SEQ ID NO. 2) was constructed, and the construction method was the same as that of the heavy chain H2 expression vector.
  • the E. coli DH5 ⁇ strain carrying the full-length genes of the heavy chain H2, H0 and light chain L0 obtained in Example 2 was cultured, the culture was harvested, and the heavy chain was extracted and purified with Qiagen UltraPure Plasmid DNA Purification Kit And the expression vector of the full-length gene of the light chain.
  • the purified plasmid DNA was transfected into 293F cells using Invitrogen's liposome method kit.
  • the transfection method refers to the kit instructions.
  • the positive cells obtained by transfection were subjected to antibody expression, and the expression level of the monoclonal antibody TCX060 with the combination of heavy chain and light chain as H2L0 is shown in Table 2.
  • the antibody H2L0 obtained by the above expression was pre-packaged with TNF- ⁇ .
  • the monoclonal antibody H2L0 was added to a 96-well plate pre-packaged with TNF- ⁇ , and the indirect method of ELISA was used to preliminarily evaluate the activity of secreted antibody to bind TNF- ⁇ .
  • the test results are shown in Table 3.
  • NC is the antibody dilution as a negative control.
  • the expression vector of the monoclonal antibody TCX060 of the heavy chain and light chain combined with H2L0 was stably transfected.
  • Transfect 293F cells by transient transfection method use Zhuhai Carrier's 293 medium and transfection reagent, collect the cell culture supernatant on the seventh day after transfection
  • the culture supernatant of the monoclonal antibody TCX060 (H2L0 combination) and the original Cytozil monoclonal antibody (H0L0) were directly separated, and purified using GE's MabSelect Sure LX.
  • the purified product is used UV spectrophotometer for quantitative detection, the calculation formula is as follows:
  • the indirect method of ELISA was used to measure antibody EC50 to evaluate antibody affinity.
  • the experimental method is as follows: Dilute TNF- ⁇ (purchased from Nearshore Technology Co., Ltd.) with PBS to the antigen to 1 ⁇ g/ml; add 100 ⁇ l/well of the diluted antigen to a 96-well plate, cover, and shake overnight at 4°C; Remove the liquid in the well, wash with PBS three times, 200ul/well, pat dry; block with 5% milk-PBS 200 ⁇ l/well for 1h, tap every 15min; shake off the liquid in the well, wash once with PBS, 200 ⁇ l/well, pat Dry; add purified antibodies (0-10 ⁇ g/ml) in gradients, see Table 4 for antibody names, diluted with 5% milk-PBS, 100 ⁇ l/well, incubate for 1h, pat every 15min; shake off the liquid in the well and wash three times with PBS , 200 ⁇ l/well, pat dry; add secondary antibody, diluted with 5% milk-PBS, 100 ⁇ l/well, incubate for 1h, pat every 15min; preheat the T
  • the CCK-8 kit was used to detect the apoptosis of mouse fibroblast cell line L929. The specific steps are as follows:
  • the control includes a negative control and a positive control.
  • the negative control only adds RPMI-1640 and cells, and the positive control only adds rhTNF ⁇ and cells.
  • the concentration of rhTNF ⁇ ranges from 2ng/ml to 8.2pg/ml.
  • the experimental results are shown in Figure 4.
  • the IC50 data is shown in Table 5.
  • the IC50 of TCX060 (H2L0) is equivalent to that of the original adamu drug, and the IC50 of TCX060 (H2L0) is lower than that of Cytozuol.
  • the immunization experiment of anti-TNF- ⁇ humanized monoclonal antibody TCX060 was performed in mice to analyze the immunogenicity of TCX060, as follows:
  • Boost immunization uses an emulsion of antigen and Freund's incomplete adjuvant.
  • the experimental method is as follows: dilute TCX060 (H2L0) or the original drug of Cytobe with PBS to 1 ⁇ g/ml; add the diluted antigen to a 96-well plate at 100 ⁇ l/well and cover it , Overnight at 4°C; shake off the liquid in the well, wash with PBS three times, 200ul/well, pat dry; block with 5% milk-PBS 200 ⁇ l/well for 1h, pat every 15min; shake off the liquid in the well and wash once with PBS , 200 ⁇ l/well, pat dry; add serum samples with different dilution ratios on different days after immunization (1:500/1:1000/1:5000/1:10000/1:50000), diluted with 5% milk-PBS, 100 ⁇ l/well, incubate for 1h, tap every 15min; shake off the liquid in the well, wash three times with PBS, 200 ⁇ l/well, pat dry; add secondary antibody, 5% milk-PBS
  • the results are shown in Figure 5.
  • the results show that the anti-TNF- ⁇ humanized monoclonal antibody TCX060 H2L0 of the present invention has a significant reduction in serum antibody titer compared with Certuzumab, so H2L0 is compared with Certuzumab Anti-, its immunogenicity has been effectively reduced.
  • the invention provides a humanized anti-TNF- ⁇ monoclonal antibody TCX060 with low immunogenicity and low ADCC/CDC function and its application.
  • the anti-TNF- ⁇ humanized monoclonal antibody TCX060 provided by the present invention is obtained by transforming the Cytozine monoclonal antibody as follows: (1) removing the PEG modification; (2) transforming into a human IgG1 subtype full-length antibody; (3) ) Insert a flexible amino acid fragment between the CDR3 and CH2 regions.
  • the binding affinity of TCX060 to human TNF- ⁇ is similar to that of Cytozhu, and it can specifically block the binding of TNF- ⁇ to cell surface TNF receptors.
  • ADCC/CDC function is similar to that of PEGylated Cytozhu monoclonal antibody, but immune The originality is significantly lower than that of the Cytozine monoclonal antibody, which can reduce the risk of immunogenicity of the antibody in the body while removing ADCC/CDC function, and has good economic value and application prospects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供一种抗TNF-α人源化单克隆抗体TCX060，所述抗体为将赛妥珠单克隆抗体经如下改造得到：(1)去除PEG修饰；(2)改造为人IgG1亚型全长抗体；(3)在CDR3和CH2区之间插入柔性氨基酸片段。

Description

低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用
交叉引用
本申请要求2019年5月7日提交的第201910376223.0号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物技术领域，具体涉及低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用。
背景技术
肿瘤坏死因子α(TNF-α)是一种由免疫细胞分泌、在炎症和免疫应答中自然分泌的细胞因子。研究显示，TNF-α在多种慢性疾病(如克罗恩氏病、多发性硬化症、类风湿性关节炎、溃疡性结肠炎等)的患者体内含量有上调的趋势(例如，在类风湿性关节炎(RA)患者的滑膜液中，TNF-α水平升高)，并在病理性炎症和关节破坏等过程中发挥重要作用。人TNF-α分子量为17kDa，在体内可以单体或三聚体形式存在，其活性形式为三聚体。TNF-α通过与细胞表面受体p55和p75相互作用以发挥生物活性功能。目前，一般采用单克隆IgG抗体或者可溶性的TNF-α受体中和体内的TNF-α。
目前市售的以TNF-α为靶点的单克隆抗体主要包括依那西普、英孚利昔、阿达木和赛妥珠等，其中依那西普是由两条人TNF-α受体(p75)偶联到Fc端的融合蛋白；英孚利昔是人源化的TNF-α鼠单克隆抗体，阿达木单克隆抗体是人源化的TNF-α抗体。利用放射性标记的TNF-α进行结合能力测定，结果表明：英孚利昔能够结合单体(无活性)及三聚体(有活性)类型的可溶TNF-α；依那西普更倾向于结合有活性的聚体形式的TNF-α及TNF-β，而阿达木单克隆抗体则只结合TNF-α，而不结合TNF-β。
由Celltech UCB公司研发的赛妥珠(Certolizumab)是一种人源化单 克隆抗体，可特异性地与TNF-α结合并阻断其与细胞表面TNF受体p55和p75的相互作用。但Certolizumab单克隆抗体在宿主体内的免疫原性较强，高达23％以上的患者使用该单克隆抗体会发生免疫反应。免疫反应可能引起免疫复合物介导的抗体或片段从循环中清除，并导致反复投药不适用于治疗，从而降低患者的治疗效益且限制抗体的再投予。同时，原研赛妥珠需要与免疫抑制剂连用，使得其产生副作用的风险大大增高。然而，目前尚没有关于低免疫原性TNF-α的抗体的报道。因此，亟需开发具有较高亲和力和特异性且具有较低的ADCC/CDC功能和免疫原性的TNF-α的抗体。
发明内容
为解决现有技术中存在的技术问题，本发明的目的在于提供低多聚体、低免疫原性、低ADCC/CDC功能的抗TNF-α人源化单克隆抗体、其制备方法及其应用。
为实现上述目的，本发明的技术方案如下：本发明通过利用商业化的DNAStarTM软件对赛妥珠单克隆抗体的氨基酸序列进行分析，结果发现未经PEG修饰的赛妥珠单克隆抗体的氨基酸原始序列的免疫原性很低，但是，经PEG化的赛妥珠单克隆抗体可能导致多聚体的产生，进而导致免疫原性提高，即PEG化虽然降低了ADCC/CDC功能，但是却导致免疫原性的问题。在上述分析的基础上，本发明在赛妥珠单克隆抗体序列的基础上改造单克隆抗体序列，以获得在维持赛妥珠较高的TNF-α结合亲和力和特异性的同时，降低其多聚体产生和免疫原性以及ADCC/CDC功能，具体改造包括：
首先，为降低赛妥珠单克隆抗体的多聚体和免疫原性，去除赛妥珠单克隆抗体的PEG修饰，然而，去除PEG修饰的抗体的ADCC/CDC功能提高；
进一步地，基于抗体诱导ADCC/CDC的作用机理，将赛妥珠单克隆抗体改造为人IgG1全长抗体。在此基础上，利用Pymol等蛋白结构分析 软件对抗体结构进行分析，在抗体可变区与恒定区之间寻找相对柔性的区域进行柔性氨基酸的插入改造；最终通过在抗体的重链CDR3区和CH2区之间插入一段柔性氨基酸片段，切断抗体可变区在结合抗原后产生的机械应力传递，使抗体的重链恒定区与Fc受体和/或补体结合位点无法充分暴露，从而弱化抗体与NK细胞、巨噬细胞和中性粒细胞等表达IgG Fc受体的杀伤细胞或补体结合，进而使得抗体无法诱导或降低诱导ADCC和CDC的信号。
本发明的第一个目的是提供一种抗TNF-α人源化单克隆抗体TCX060，其为将Certolizumab单克隆抗体经如下改造得到：
(1)去除PEG修饰；
(2)通过添加经改造的人IgG1亚型抗体的重链恒定区改造为人IgG1亚型全长抗体；
(3)在得到的人IgG1亚型全长抗体的重链CDR3和CH2区之间插入柔性氨基酸片段；
所述Certolizumab单克隆抗体的轻链全长序列和重链可变区序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示；
所述抗TNF-α人源化单克隆抗体TCX060具有结合人TNF-α并阻断人TNF-α与TNF受体结合的功能。
上述步骤(3)中，在得到的人IgG1亚型全长抗体的CDR3和CH2区之间插入柔性氨基酸片段，可以阻断抗体可变区同恒定区之间的应力传递，使得抗体的Fc受体和/或补体结合位点无法充分暴露，进而有效降低抗体的ADCC/CDC效应。
为能够更好地阻断抗体可变区在结合抗原后产生的机械应力传递，作为优选，所述柔性氨基酸片段的插入位置为如SEQ ID NO.3所示序列的第237位与第238位氨基酸之间。所述柔性氨基酸片段含有一个或多个甘氨酸或丝氨酸。
进一步优选地，所述柔性氨基酸片段的序列为GGGS、GGSGGS或 GSGSGS。
作为本发明的一种优选实施方式，所述柔性氨基酸片段的插入位置为如SEQ ID NO.3所示序列的第237位与第238位氨基酸之间；所述柔性氨基酸片段的序列为GGGS。
本发明对基于上述改造得到的抗TNF-α人源化单克隆抗体的重链进行大量筛选，获得了在免疫原性和ADCC/CDC功能方面性能最优的抗TNF-α人源化单克隆抗体的重链，其全长序列如SEQ ID NO.4所示或为如SEQ ID NO.4所示的氨基酸序列经一个或多个氨基酸的替换、缺失或插入得到的具有相同功能多肽的氨基酸序列。
本发明中，上述“经一个或多个氨基酸的替换、缺失或插入得到的具有相同功能的蛋白的氨基酸序列”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列，其可为具有如SEQ ID NO.4所示的氨基酸序列的重链全长的“保守修饰的变体”或经“保守的氨基酸取代”改造得到的，“保守修饰的变体”或经“保守的氨基酸取代”是指本领域技术人员已知的氨基酸取代，进行这种取代通常不改变所得到的分子的生物学活性。一般而言，本领域技术人员公认在多肽非必需区的单个氨基酸取代基本上不改变生物学活性。示例性取代优选依照表1所示的取代进行：
表1示例性保守氨基酸取代表
原残基 保守取代
Ala(A) Gly,Ser
Arg(R) Lys,His
Asn(N) Gln,His
Asp(D) Glu,Asn
Cys(C) Ser,Ala
Gln(Q) Asn
Glu(E) Asp,Gln
Gly(G) Ala
His(H) Asn,Gln
Ile(I) Leu,Val
Lys(K) Arg,His
Met(M) Leu,Ile,Tyr
Phe(F) Tyr,Met,Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr,Phe
Tyr(Y) Trp,Phe
Val(V) Ile,Leu
上述序列如SEQ ID NO.4所示的重链可以与经过改造或未经改造的Certolizumab单克隆抗体的轻链组合，有效降低未经PEG化的Certolizumab单克隆抗体的ADCC/CDC功能，同时具有较低的免疫原性。
通过对具备上述轻链和重链全长序列的单克隆抗体进行筛选，本发明得到1个最优的轻链和重链全长组合构成的人TNF-α单克隆抗体TCX060，其重链全长氨基酸序列如SEQ ID NO.4所示，轻链全长氨基酸序列如SEQ ID NO.1所示，该单克隆抗体保留了Certolizumab抗体的抗原结合位点，与Certolizumab抗体具有相近的表达量，并且与人TNF-α结合的亲和力与Certolizumab抗体相近，可特异性地阻断TNF-α与细胞表面TNF受体p55和p75的结合；其ADCC/CDC功能与PEG化的Certolizumab抗体相近，但与Certolizumab抗体相比，该单克隆抗体具有更低的免疫原性。
本发明的第二个目的在于提供编码所述抗TNF-α人源化单克隆抗体TCX060的基因。
编码抗TNF-α人源化单克隆抗体TCX060的基因包括任何编码本发明提供的抗TNF-α人源化单克隆抗体的重链或轻链的核酸；根据密码子的简并性，其可以为根据任何宿主密码子偏好性进行密码子优化获得的编码所述轻链和重链全长氨基酸序列的核酸。
作为优选，上述基因中，编码重链全长的核苷酸序列如SEQ ID NO.5所示，编码轻链全长的核苷酸序列如SEQ ID NO.6所示。
本发明的第三个目的在于提供包含编码所述抗TNF-α人源化单克隆抗体TCX060的基因的生物材料。
所述生物材料包括表达盒、载体、宿主细胞、工程菌或细胞系。
本发明的第四个目的在于提供所述抗TNF-α人源化单克隆抗体TCX060的制备方法，其通过表达所述编码重链全长和轻链全长的基因实现。
作为本发明的一种实施方式，所述抗TNF-α人源化单克隆抗体TCX060的制备方法包括如下步骤：
(1)合成所述抗TNF-α人源化单克隆抗体的重链全长基因和轻链全长基因；
(2)将上述合成基因连接至表达载体；
(3)将连接有上述抗TNF-α人源化单克隆抗体的重链全长基因和轻链全长基因的表达载体转化至宿主细胞；
(4)筛选获得稳定表达所述抗TNF-α人源化单克隆抗体的宿主细胞，培养所述宿主细胞，表达抗TNF-α人源化单克隆抗体；
(5)提取纯化获得抗TNF-α人源化单克隆抗体。
本发明的第五个目的在于提供所述抗TNF-α人源化单克隆抗体TCX060或编码所述抗TNF-α人源化单克隆抗体TCX060基因或含有所述基因的生物材料在制备以人TNF-α为靶标的药物中的应用。
优选地，所述以人TNF-α为靶标的药物为预防或治疗肿瘤、炎症或自身免疫性疾病的药物。
所述的疾病包括但不限于克罗恩病、类风湿关节炎、银屑病关节炎、活动性强直性脊柱炎等。
本发明的第六个目的在于提供所述抗TNF-α人源化单克隆抗体TCX060或编码所述抗TNF-α人源化单克隆抗体TCX060基因或含有所述基因的生物材料在制备人TNF-α检测试剂中的应用。
本发明的第七个目的在于提供包含所述抗TNF-α人源化单克隆抗体TCX060的药物或检测试剂。
所述包含所述抗TNF-α人源化单克隆抗体TCX060的药物还可以包 含其它药学领域允许的有效成分或辅料。
本发明的有益效果在于：
本发明在赛妥珠单克隆抗体的基础上通过人工改造获得抗TNF-α人源化单克隆抗体，本发明提供的人源化抗TNF-α单克隆抗体与赛妥珠单克隆抗体具有相同的TNF-α抗原结合位点，但其抗体构象以及免疫原性等性质与赛妥珠不同。本发明提供的人源化抗TNF-α单克隆抗体TCX060保留了赛妥珠单克隆抗体的抗原亲和力和特异性，且其诱导ADCC/CDC功能与PEG化的赛妥珠单克隆抗体相近，但其免疫原性显著低于赛妥珠单克隆抗体；同时，本发明提供的人源化抗TNF-α单克隆抗体的生产工艺更加简化。更低的免疫原性将降低人体内由抗体免疫原性引起的免疫反应导致的药物副作用风险，同时，免疫原性的降低一方面延长了由本发明提供的人源化抗TNF-α单克隆抗体制备的抗体药的体内半衰期，更大程度地避免了抗体药ADA引起的药效降低；一方面可减少抗体药用量从而降低治疗成本；另一方面可摆脱抗TNF-α单克隆抗体药与免疫抑制剂联合给药的模式，实现抗TNF-α单克隆抗体药单独给药的效果，去除与免疫抑制剂联用所带来的副作用，提高用药安全性。本发明提供的人源化抗TNF-α单克隆抗体TCX060具有极大的应用潜力和价值，有望成为理想的生物靶向治疗抗体。
附图说明
图1为本发明实施例2中赛妥珠单克隆抗体编码基因及本发明提供的H2L0单克隆抗体编码基因的表达载体双酶切鉴定电泳结果图，其中，a为赛妥珠单克隆抗体的轻链L0质粒Hind III和EcoRⅠ双酶切结果，泳道从左到右分别为：酶切前质粒、酶切后质粒及DNA marker；b为本发明提供的单克隆抗体TCX060的重链H2质粒Hind III和EcoRⅠ双酶切结果，泳道从左到右分别为：酶切前质粒、酶切后质粒及DNA marker。
图2为本发明实施例4中单克隆抗体TCX060的SDS-PAGE电泳图，泳道从左到右依次为：表达上清、Marker、纯化抗体。
图3为本发明实施例5中单克隆抗体TCX060的亲和力EC 50；其中H2L0代表TCX060。
图4为本发明实施例5中单克隆抗体TCX060的TNF-α介导的细胞杀伤实验结果；其中，Adalimumab代表阿达木原研药，Certolizumab代表赛妥珠原研药，H2L0代表本发明抗体TCX060H2L0。
图5为本发明实施例6中单克隆抗体TCX060的小鼠ADA评价结果；其中，Certolizumab Day 0、Certolizumab Day 7、Certolizumab Day 14分别代表赛妥珠原研药免疫前，免疫后第7，第14天血清样本，H2L0Day0、H2L0Day 7、H2L0Day 14分别代表本发明抗体药物TCX060H2L0免疫前，免疫后第7，第14天血清样本。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1低ADCC/CDC功能的降低免疫原性的Certolizumab单克隆抗体的分析及设计
本发明通过利用商业化的DNAStarTM软件对赛妥珠单克隆抗体的原始序列进行分析评价，分析结果显示，原研赛妥珠单克隆抗体序列的免疫原性很低。由此推测，在原研赛妥珠单克隆抗体进行为去除ADCC/CDC功能而进行的PEG化后，使得抗体的多聚体增加，进而导致了药物的ADA高达23％。因此，需要对原研赛妥珠单克隆抗体进行去除PEG化修饰，同时降低其ADCC/CDC功能。原始赛妥珠的轻链全长和重链可变区的氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
首先对原研赛妥珠单克隆抗体去除PEG修饰，并进行人IgG1亚型全抗体改造，在此基础上，通过在改造获得的人IgG1亚型全抗体的可变区与恒定区之间寻找相对柔性的区域，通过在柔性区域插入柔性氨基酸序列，切断抗体可变区结合抗原后产生的机械应力传递，使得抗体重链恒定区与Fc受体和/或补体结合位点不能充分暴露出来，降低抗体与NK细胞、巨噬细胞和中性粒细胞等表达IgG Fc受体的杀伤细胞结合或和补体的结合，使得抗体无法诱导或减弱诱导ADCC和CDC的信号。通过Pymol软件分析确定在上述赛妥珠人IgG1亚型全抗体的重链CDR3与CH2区之间插入一段柔性氨基酸序列可以更好地阻断抗体结合抗原后抗体可变区及恒定区间的应力传递，在降低ADCC/CDC功能的同时不会显著增加抗体多聚体的形成。通过大量的序列分析、结构模拟预测，确定了重链序列如SEQ ID NO.3所示的赛妥珠人IgG1亚型全抗体，并确定通过在如SEQ ID NO.3所示序列的第237位与第238位氨基酸之间插入柔性氨基酸序列GGGS，得到改造后的人IgG1亚型全抗体的重链H2，其序列如SEQ ID NO.4所示；通过对不同的轻链和重链组合得到的单克隆抗体的综合性能进行初步评价，最终得到由轻链L0(赛妥珠原始轻链序列，SEQ ID NO.1)和重链H2(SEQ ID NO.4)组成的抗TNF-α人源化单克隆抗体H2L0。将该单克隆抗体命名为TCX060。
实施例2抗TNF-α人源化单克隆抗体TCX060的表达载体的构建
根据实施例1得到的抗TNF-α全人源化单克隆抗体H2L0的重链和轻链全长的氨基酸序列以及宿主的密码子偏好性，设计编码重链H2和轻链L0的核苷酸序列(其中L0的核苷酸序列如SEQ ID NO.6所示，H2的核苷酸序列如SEQ ID NO.5所示)，设计轻链序列两侧的酶切位点为Hind III+EcoR I，设计重链序列两侧的酶切位点为Hind III+EcoR I，将携带酶切位点的重链全长和轻链全长序列送交金唯智公司合成全基因序列，合成所用的连接载体为pUC57。以pEE12.4(用于重链表达)及pEE6.4(用于轻链表达)为表达载体，将上述表达载体与合成的基因序列分别进行相应的 双酶切，将酶切得到的目的基因和表达载体分别进行琼脂糖凝胶电泳分离，随后切下目的条带并用Qiagen Gel Extraction Kit回收，按T4DNA酶连接体系，16℃过夜连接，然后转化大肠杆菌DH5α，对得到的转化子进行PCR鉴定和质粒提取、序列测定，得到分别携带重链H2和轻链L0全长基因的表达载体。以原研赛妥珠作为对照，构建携带原研赛妥珠重链H0(序列如SEQ ID NO.2所示)的pEE12.4表达载体，构建方法与重链H2的表达载体相同。
携带上述重链和轻链全长基因的表达载体的双酶切鉴定电泳结果如图1所示。
实施例3抗TNF-α人源化单克隆抗体TCX060的瞬时表达
将实施例2获得的分别携带重链H2、H0和轻链L0的全长基因的表达载体的大肠杆菌DH5α菌株进行培养，收获培养物，用Qiagen UltraPure质粒DNA纯化试剂盒抽提纯化携带重链和轻链全长基因的表达载体。将上述纯化的质粒DNA采用Invitrogen公司的脂质体法试剂盒转染293F细胞，转染方法参照试剂盒说明书。
将转染获得的阳性细胞进行抗体的表达，重链轻链组合为H2L0的单克隆抗体TCX060的表达量如表2所示。
表2单克隆抗体TCX060的表达量检测结果(mg/L)
Figure PCTCN2020088253-appb-000001
对上述表达获得的抗体H2L0进行预包TNF-α实验，将单克隆抗体H2L0加入预包有TNF-α的96孔板中，采用ELISA间接法，初步评价分泌抗体结合TNF-α的活性。检测结果如表3所示。NC为以抗体稀释液作为阴性对照。
表3单克隆抗体TCX060的活性评价结果
  赛妥珠原研药 H2L0
EC50(M) 9.374e-011 2.464e-011
表3结果显示，H2L0组合所对应的单克隆抗体TCX060得到了表达，表3结果表明，单克隆抗体TCX060可识别TNF-α。
实施例4抗TNF-α人源化单克隆抗体TCX060的表达与纯化
根据实施例3的检测结果，将重链轻链组合H2L0的单克隆抗体TCX060的表达载体进行稳定转染。
采用瞬时转染方法转染293F细胞，使用珠海恺瑞公司的293培养基及转染试剂，于转染后第七天收集细胞培养上清
将单克隆抗体TCX060(H2L0组合)及原研赛妥珠单克隆抗体(H0L0)的培养上清直接分离，并利用GE公司的MabSelect Sure LX进行纯化，实验过程详见UCB公司说明书，纯化后产品利用紫外分光光度计进行定量检测，计算公式如下：
浓度计算：将收集的洗脱峰读OD 280后，计算浓度。抗体浓度＝OD 280/1.4。
抗体纯化后进行SDS-PAGE电泳验证，结果如图2所示，电泳结果表明，纯化抗体的纯度超过80％以上，且条带相对单一，浓度相当，可进行后期的实验。
实施例5抗TNF-α人源化单克隆抗体TCX060的生物活性测定
1、亲和力评价
本实施例利用ELISA间接法测抗体EC50评价抗体亲和力。
实验方法如下：用PBS将TNF-α(购自近岸科技有限公司)稀释抗原到1μg/ml；将稀释好的抗原按100μl/孔加到96孔板中，加盖，4℃过夜；甩去孔内液体，用PBS洗三次，200ul/孔，拍干；用5％牛乳-PBS 200μl/孔封闭1h，每15min轻拍；甩去孔内液体，用PBS洗一次，200μl/孔，拍干；分梯度加入纯化抗体(0-10μg/ml)，抗体名称见表4，5％牛乳-PBS稀释，100μl/孔，孵育1h，每15min轻拍；甩去孔内液体，用PBS洗三次，200μl/孔，拍干；加二抗，5％牛乳-PBS稀释，100μl/孔，孵育1h，每15min轻拍；预热TMB底物于室温，同时开启酶标仪预热；PBS洗5 次，250μl/well，前三次5min，后两次10min，拍干；TMB底物A、B各加50μl/孔，室温显色20min；使用扫描仪将图片扫描记录，加入终止液50μl/孔，使用酶标仪读取OD 450。实验数据见表4和图3。
表4单克隆抗体TCX060的EC50检测结果
Figure PCTCN2020088253-appb-000002
以上结果表明：单克隆抗体TCX060对人TNF-α的亲和力较原研赛妥珠对人TNF-α的亲和力略高。
2、细胞毒作用实验
利用CCK-8试剂盒对小鼠成纤维细胞株L929进行细胞凋亡的检测。具体操作步骤如下：
在96孔板中加入100μl三倍梯度稀释的单克隆抗体(RPMI-1640培养基含10％FBS稀释)(单克隆抗体TCX060的加入浓度依照FDA中提及的关于赛妥珠的该实验的IC50分别设置上下3-5个梯度浓度)，随后加入50μl终浓度为500pg/ml的rhTNF-α(RPMI-1640培养基含10％FBS稀释)，室温孵育30min。向每孔加入50μl L929细胞5×10 4/孔,包含终浓度为1μg/ml的放线菌素-D。37℃培养箱孵育过夜培养(18-24h)。对照包括阴性对照和阳性对照，阴性对照只加入RPMI-1640及细胞，阳性对照只加入rhTNFα及细胞，rhTNFα浓度从2ng/ml到8.2pg/ml梯度。向每孔加入20μL CCK8溶液(注意不要在孔中生成气泡，以免影响OD值的读数)。将培养板在37℃培养箱内孵育1-4h。用酶标仪测定在450nm处的吸光度。若暂时不测定OD值，可以向每孔中加入10μL 0.1M的HCL溶液或者1％w/v SDS溶液，并遮盖培养板避光保存在室温条件下。24小时内测定，吸光度不会发生变化。
计算抗体在不同浓度下的细胞杀伤率和IC50。
计算公式：(单克隆抗体TCX060处理细胞-TNF-α处理细胞)/TNF-α处理细胞×100％。
实验结果见图4。IC50数据如表5所示，TCX060(H2L0)的IC50与阿达木原研药相当，TCX060(H2L0)的IC50与赛妥珠相比更低。
表5单克隆抗体TCX060的IC50检测结果
  TA001(阿达木原研药) Q002(赛妥珠) TCX060H2L0
IC50(M) 1.478e-008 1.305e-006 ～4.249e-008
实施例6抗TNF-α人源化单克隆抗体TCX060的免疫原性评价
在小鼠体内进行抗TNF-α人源化单克隆抗体TCX060的免疫实验，分析TCX060的免疫原性，具体如下：
1)基础免疫：将TCX060(H2L0)和赛妥珠原研药与福氏完全佐剂等体积混合并充分乳化，分点皮下注射，每只Balb/c小鼠每次注射量为70μg。
2)加强免疫：加强免疫采用抗原与福氏不完全佐剂的乳化液。
上述实验完成后进行ELISA检测试验，实验方法如下：用PBS将TCX060(H2L0)或赛妥珠原研药稀释到1μg/ml；将稀释好的抗原按100μl/孔加到96孔板中，加盖，4℃过夜；甩去孔内液体，用PBS洗三次，200ul/孔，拍干；用5％牛乳-PBS 200μl/孔封闭1h，每15min轻拍；甩去孔内液体，用PBS洗一次，200μl/孔，拍干；分梯度加入免疫后不同天不同稀释比例的血清样本(1:500/1:1000/1:5000/1:10000/1:50000)，5％牛乳-PBS稀释，100μl/孔，孵育1h，每15min轻拍；甩去孔内液体，用PBS洗三次，200μl/孔，拍干；加二抗，5％牛乳-PBS稀释，100μl/孔，孵育1h，每15min轻拍；预热TMB底物于室温，同时开启酶标仪预热；PBS洗5次，250μl/well，前三次5min，后两次10min，拍干；TMB底物A、B各加50μl/孔，室温显色20min；使用扫描仪将图片扫描记录，加入终止液50μl/孔，使用酶标仪读取OD 450。
结果见图5，结果表明，本发明的抗TNF-α人源化单克隆抗体TCX060 H2L0同赛妥珠单抗相比，血清中的抗体滴度显著降低，因此H2L0相比于赛妥珠单抗，其免疫原性得到了有效的降低。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用。本发明提供的抗TNF-α人源化单克隆抗体TCX060，为将赛妥珠单克隆抗体经如下改造得到：(1)去除PEG修饰；(2)改造为人IgG1亚型全长抗体；(3)在CDR3和CH2区之间插入柔性氨基酸片段。TCX060与人TNF-α结合的亲和力与赛妥珠相近，可特异性地阻断TNF-α与细胞表面TNF受体结合，其ADCC/CDC功能与PEG化赛妥珠单克隆抗体相近，但免疫原性显著低于赛妥珠单克隆抗体，在去除ADCC/CDC功能的同时减少抗体在体内产生免疫原性的风险，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种抗TNF-α人源化单克隆抗体TCX060，其特征在于，其为将Certolizumab单克隆抗体经如下改造得到：
    (1)去除PEG修饰；
    (2)通过添加经改造的人IgG1亚型抗体的重链恒定区改造为人IgG1亚型全长抗体；
    (3)在得到的人IgG1亚型全长抗体的重链CDR3和CH2区之间插入柔性氨基酸片段；
    所述Certolizumab单克隆抗体的轻链全长序列和重链可变区序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示；
    所述抗TNF-α人源化单克隆抗体TCX060具有结合人TNF-α并阻断人TNF-α与TNF受体结合的功能。
  2. 根据权利要求1所述的单克隆抗体TCX060，其特征在于，所述柔性氨基酸片段的插入位置为如SEQ ID NO.3所示序列的第237位与第238位氨基酸之间；所述柔性氨基酸片段含有一个或多个甘氨酸和丝氨酸。
  3. 根据权利要求2所述的单克隆抗体TCX060，其特征在于，所述柔性氨基酸片段的序列为选自GGGS、GGSGGS、GSGSGS中的一种。
  4. 根据权利要求1～3任一项所述的单克隆抗体TCX060，其特征在于，所述单克隆抗体TCX060的重链全长序列如SEQ ID NO.4所示或为如SEQ ID NO.4所示的氨基酸序列经一个或多个氨基酸的替换、缺失或插入得到的具有相同功能多肽的氨基酸序列。
  5. 编码权利要求1～4任一项所述单克隆抗体TCX060的基因。
  6. 根据权利要求5所述的基因，其特征在于，编码重链全长的核苷酸序列如SEQ ID NO.5所示，编码轻链全长的核苷酸序列如SEQ ID NO.6所示。
  7. 包含权利要求5或6所述基因的生物材料，其特征在于，所述生物材料包括表达盒、载体、宿主细胞、工程菌或细胞系。
  8. 权利要求1～4任一项所述单克隆抗体TCX060或权利要求5或6所述基因或权利要求7所述生物材料在制备以人TNF-α为靶标的药物中的应用；
    优选地，所述以人TNF-α为靶标的药物为预防或治疗肿瘤、炎症或自身免疫性疾病的药物。
  9. 权利要求1～4任一项所述单克隆抗体TCX060或权利要求5或6所述基因或权利要求7所述生物材料在制备人TNF-α检测试剂中的应用。
  10. 包含权利要求1～4任一项所述单克隆抗体TCX060的药物或检测试剂。
PCT/CN2020/088253 2019-05-07 2020-04-30 低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用 WO2020224529A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2020270242A AU2020270242B2 (en) 2019-05-07 2020-04-30 Anti-TNF-α humanized monoclonal antibody TCX060 having low immunogenicity and low ADCC/CDC function, and use thereof
US17/609,778 US20230242634A1 (en) 2019-05-07 2020-04-30 Anti-tnf-alpha humanized monoclonal antibody tcx060 having low immunogenicity and low adcc/cdc function, and use thereof
EP20801746.7A EP3967708A4 (en) 2019-05-07 2020-04-30 HUMANIZED MONOCLONAL ANTI-TNF-ALPHA ANTIBODY TCX060 WITH LOW IMMUNOGENICITY AND LOW ADCC/CDC FUNCTION AND ITS USE
JP2021566946A JP7300008B2 (ja) 2019-05-07 2020-04-30 低免疫原性かつ低ＡＤＣＣ／ＣＤＣ機能の抗ＴＮＦ－αヒト化モノクローナル抗体ＴＣＸ０６０およびその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910376223 2019-05-07
CN201910376223.0 2019-05-07

Publications (1)

Publication Number Publication Date
WO2020224529A1 true WO2020224529A1 (zh) 2020-11-12

Family

ID=73050711

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/088253 WO2020224529A1 (zh) 2019-05-07 2020-04-30 低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用

Country Status (6)

Country Link
US (1) US20230242634A1 (zh)
EP (1) EP3967708A4 (zh)
JP (1) JP7300008B2 (zh)
CN (1) CN111909268B (zh)
AU (1) AU2020270242B2 (zh)
WO (1) WO2020224529A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116903738A (zh) * 2022-08-02 2023-10-20 北京绿竹生物技术股份有限公司 一种低甘露糖型抗人肿瘤坏死因子-α单抗及其用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1383450A (zh) * 2000-06-06 2002-12-04 细胞技术研究及开发有限公司 对人肿瘤坏死因子α具有特异性的抗体分子及其用途
CN1822857A (zh) * 2003-06-02 2006-08-23 阿莱克申药物公司 去免疫原性抗cd3抗体
CN101426519A (zh) * 2004-03-31 2009-05-06 森托科尔公司 人glp-1模拟体、组合物、方法和用途
CN104788565A (zh) * 2003-05-02 2015-07-22 赞科股份有限公司 优化的Fc变体及其产生方法
CN107207582A (zh) * 2015-02-16 2017-09-26 隆萨有限公司 用于药物缀合的cl和/或ch1突变抗体
CN107949570A (zh) * 2015-06-30 2018-04-20 勃林格殷格翰国际有限公司 多特异性结合蛋白

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UA86605C2 (ru) * 2004-01-12 2009-05-12 Аплайд Молекьюлер Иволюшн, Инк. Антитело, которое содержит вариант исходного человеческого fс-участка
BRPI0710011A2 (pt) * 2006-04-14 2011-08-02 Trubion Pharmaceuticals Inc proteìnas de ligação compreendendo articulação de imunoglobulina e regiões fc dotadas de funções efetoras fc alteradas
CA2756988A1 (en) * 2009-04-01 2010-10-07 Genentech, Inc. Anti-fcrh5 antibodies and immunoconjugates
RU2595379C2 (ru) * 2009-04-16 2016-08-27 АббВай Биотерапеутикс Инк. АНТИТЕЛА ПРОТИВ TNF-α И ИХ ПРИМЕНЕНИЯ
EP2791170A1 (en) * 2011-12-16 2014-10-22 Synthon Biopharmaceuticals B.V. Compounds and methods for treating inflammatory diseases
CN104341502B (zh) * 2013-08-09 2016-04-27 北京天成新脉生物技术有限公司 低免疫原性抗TNF-α全人源单抗及其应用
CR20180365A (es) * 2015-12-16 2018-09-28 Amgen Inc PROTEÍNAS DE UNIÓN AL ANTÍGENO BISPECÍFICO DE ANTI-TL1A/ANTI-TNF-a Y SUS USOS
US20200040084A1 (en) * 2017-01-06 2020-02-06 Momenta Pharmaceuticals, Inc. Compositions and methods related to engineered fc-antigen binding domain constructs

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1383450A (zh) * 2000-06-06 2002-12-04 细胞技术研究及开发有限公司 对人肿瘤坏死因子α具有特异性的抗体分子及其用途
CN104788565A (zh) * 2003-05-02 2015-07-22 赞科股份有限公司 优化的Fc变体及其产生方法
CN1822857A (zh) * 2003-06-02 2006-08-23 阿莱克申药物公司 去免疫原性抗cd3抗体
CN101426519A (zh) * 2004-03-31 2009-05-06 森托科尔公司 人glp-1模拟体、组合物、方法和用途
CN107207582A (zh) * 2015-02-16 2017-09-26 隆萨有限公司 用于药物缀合的cl和/或ch1突变抗体
CN107949570A (zh) * 2015-06-30 2018-04-20 勃林格殷格翰国际有限公司 多特异性结合蛋白

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE, J. H. ET AL.: "Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells", PROTEIN EXPRESSION PURIFICATION, vol. 87, no. 1, 31 October 2013 (2013-10-31), XP055333326, ISSN: 1046-5928, DOI: 20200613154001A *
See also references of EP3967708A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116903738A (zh) * 2022-08-02 2023-10-20 北京绿竹生物技术股份有限公司 一种低甘露糖型抗人肿瘤坏死因子-α单抗及其用途

Also Published As

Publication number Publication date
EP3967708A1 (en) 2022-03-16
JP7300008B2 (ja) 2023-06-28
EP3967708A4 (en) 2023-01-04
AU2020270242A1 (en) 2022-01-06
CN111909268B (zh) 2022-04-19
JP2022532582A (ja) 2022-07-15
US20230242634A1 (en) 2023-08-03
AU2020270242B2 (en) 2024-06-06
CN111909268A (zh) 2020-11-10

Similar Documents

Publication Publication Date Title
EP0617126B1 (en) Polypeptide capable of inhibiting the binding between human IL-6 and its receptor
WO2020186974A1 (zh) 一种双特异性抗体及其制备方法与应用
CN108409860B (zh) 抗人白细胞介素-4受体α单克隆抗体、其制备方法和应用
CN106336459B (zh) 抗人白细胞介素-17a单克隆抗体、其制备方法和应用
JPH08507201A (ja) リンホトキシン‐β、リンホトキシン‐β複合体、それらの薬学的な調製物および治療への使用
US11667704B2 (en) Anti-IL-17 antibody/TNFR ECD fusion protein and use thereof
WO2016019726A1 (zh) 低免疫原性抗TNF-α全人源单抗及其应用
CN114380906B (zh) 一种抗il-17a的单域抗体及其用途
JP2014124186A (ja) 多価抗体フラグメントおよびその三量体化複合体
WO2020224529A1 (zh) 低免疫原性低ADCC/CDC功能抗TNF-α人源化单克隆抗体TCX060及其应用
CN117940461A (zh) 一种双特异性抗体及其用途
WO2020108463A1 (zh) 人源化抗人ox40单克隆抗体及其制备方法和用途
CN105916883B (zh) 双功能融合蛋白及其制备方法和用途
CN114591432B (zh) 抗TNFα的单域抗体及其用途
WO2010105446A1 (zh) 抗人肿瘤坏死因子受体α单抗及其应用
CN106540252B (zh) 针对肿瘤坏死因子α的蛋白疫苗及其用途
WO2020224530A1 (zh) 低免疫原性抗TNF-α人源化单克隆抗体TCX063及其应用
US8895237B2 (en) Enhancing of hepatitis B virus vaccine and its gene
Zhan et al. Fusion of HSA influences TNF-α neutralizing activity of shTNFRs
WO2016070769A1 (zh) 同时阻断b7/cd28和il6/il6r/gp130信号通路的重组融合蛋白
WO2011085574A1 (zh) 全人TNFα单克隆抗体及其制备方法与应用
US11673948B2 (en) Anti-TNF/IFN scFv-Fc bispecific antibody and uses thereof
CN109400709B (zh) 双功能抗体及其用途
WO2024125445A1 (zh) 一种靶向TNF-α与IL-17A的双特异性融合蛋白及其用途
CN102336834B (zh) 全人TNFα-Fab抗体及其PEG化抗体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20801746

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021566946

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020801746

Country of ref document: EP

Effective date: 20211207

ENP Entry into the national phase

Ref document number: 2020270242

Country of ref document: AU

Date of ref document: 20200430

Kind code of ref document: A