WO2016112497A1 - Anticorps humain anti-interféron alpha humain et son application - Google Patents

Anticorps humain anti-interféron alpha humain et son application Download PDF

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WO2016112497A1
WO2016112497A1 PCT/CN2015/070625 CN2015070625W WO2016112497A1 WO 2016112497 A1 WO2016112497 A1 WO 2016112497A1 CN 2015070625 W CN2015070625 W CN 2015070625W WO 2016112497 A1 WO2016112497 A1 WO 2016112497A1
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antibody
ifnα
seq
sequence
variable region
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PCT/CN2015/070625
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Chinese (zh)
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孙志伟
王双
杜鹏
仇炜祎
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中国人民解放军军事医学科学院生物工程研究所
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Priority to PCT/CN2015/070625 priority Critical patent/WO2016112497A1/fr
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention relates to the preparation and application of a human genetically engineered antibody for treatment, which is mainly for specifically targeting a plurality of subtypes of human interferon alpha (IFN ⁇ ), and inhibiting the biology of IFN ⁇ by blocking the binding of IFN ⁇ and IFN ⁇ receptors.
  • a human genetically engineered antibody for treatment which is mainly for specifically targeting a plurality of subtypes of human interferon alpha (IFN ⁇ ), and inhibiting the biology of IFN ⁇ by blocking the binding of IFN ⁇ and IFN ⁇ receptors.
  • Interferon is a glycoprotein produced by a virus or other interferon-inducing agent that stimulates the reticuloendothelial system, macrophages, lymphocytes, and somatic cells.
  • Interferon is a broad-spectrum antiviral agent that does not directly kill or inhibit the virus, but mainly causes cells to produce antiviral proteins through cell surface receptors, thereby inhibiting virus replication; and also enhancing natural killer cells (NK)
  • NK natural killer cells
  • the vitality of cells, macrophages and T lymphocytes thereby playing an immunomodulatory role and enhancing antiviral capacity. They have a broad spectrum of antiviral, antiproliferative, immunomodulatory and induced differentiation effects on a variety of cells.
  • interferon has the following characteristics: 1 indirect; 2 broad-spectrum; 3 gene-specific: generally high activity in the same species of cells, inactive to xenogeneic cells; 4 rapid effect: interferon can Disruption of viral infection of infected cells can limit the spread of the virus; interferon plays an important role before the initial stage of infection, humoral immunity and cellular immunity (Lu Zaiying, Zhong Nanshan, et al., Internal Medicine, 7th ed.). As an important cytokine drug, interferon plays a huge role in the treatment of various malignant tumors and the prevention and treatment of viral diseases.
  • Interferon is divided into type I, type II and type III.
  • There are 7 types of interferon type I: IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ humans do not have IFN- ⁇ and IFN- ⁇ ; IFN- ⁇ (and only one subtype), type III interferons have IFN- ⁇ (at least three subtypes are currently found).
  • IFN- ⁇ and only one subtype
  • type III interferons have IFN- ⁇ (at least three subtypes are currently found).
  • there are about 13 subtypes of IFN- ⁇ in type I interferons and some subtypes can be further subdivided according to the difference in alleles. Different types of type I interferons and their typing have different specific activities, but their biological functions are consistent.
  • IFNAR interferon receptor
  • IFNAR2 contains both IFNAR1 and IFNAR2 chains and is a glycoprotein.
  • IFNAR2 has three different splicing forms: long transmembrane receptor (IFNAR2c), short transmembrane receptor (IFNAR2b) and soluble receptor (IFNAR2a).
  • Type I interferons produce a series of biological responses by binding to IFNAR2c alone or IFNAR1 to the IFNAR2c complex membrane receptor, activating the downstream JAK-STAT pathway.
  • type I interferon can promote the differentiation of naive T cells into Th1; maintain the survival and functional viability of memory T cells; promote the differentiation of plasmablasts into plasma cells, enhance antibody production; induce/activate dendritic cells Form and so on.
  • type I interferon can promote the differentiation of naive T cells into Th1; maintain the survival and functional viability of memory T cells; promote the differentiation of plasmablasts into plasma cells, enhance antibody production; induce/activate dendritic cells Form and so on.
  • a large number Studies have shown that a variety of autoimmune diseases may be associated with high expression of type I interferons.
  • IFN ⁇ insulin-dependent diabetes mellitus
  • SLE systemic lupus erythematosus
  • autoimmune thyroiditis closely related.
  • IFN ⁇ activity may be beneficial for a variety of autoimmune patients.
  • Sifalimumab from MedImmune and Rontalizumab from Genetech have entered the phase II clinical trial of SLE for psoriasis, dermatomyositis, polymyositis and so on. Clinical studies of immunological treatment are also underway.
  • Human antibodies are the ultimate direction for the development of therapeutic antibodies.
  • Clinical application of human antibodies especially in the treatment of autoimmune diseases, can minimize the immunogenicity of antibodies, prolong the half-life of drugs in vivo and enable human immunoglobulins.
  • the Fc segment of the protein mediates effects such as immunomodulation, ADCC and CDC, thereby enhancing the biological effects of the antibody.
  • the main development tools for human antibodies include antibody library technology and transgenic mouse technology. Among them, antibody library technology is widely favored by antibody developers because of its simplicity, convenience and flexibility. At present, this technology is very mature and is considered to be one of the most successful technical methods for developing therapeutic antibodies.
  • a first object of the present invention is to provide an amino acid sequence of a human anti-IFN ⁇ antibody and an active fragment thereof.
  • a second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.
  • a third object of the present invention is to provide an application of the above antibody and an active fragment thereof for the preparation of a medicament for treating an IFN ⁇ -related autoimmune disease.
  • the antibody variable region amino acid sequence pattern provided by the present invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • FR1 to 4 represent four frame regions, and CDRs 1 to 3 represent three hypervariable regions.
  • FR1 ⁇ 4 may be isolated from the constant region sequence (such as the most commonly used amino acids of human immunoglobulin light heavy chain, subclass or subfamily), or may be isolated from the individual antibody framework regions or from different framework regions.
  • the genes come together. For example, a number of human antibody framework region sequences included in the library of Kabat et al. Among them, the heavy chain is the human immunoglobulin subgroup human heavy chain VH III family, and the light chain is the Lambda I family.
  • CDR1L sequence was: SGSSSNX1GSNX2VX3 (SEQ ID NO 1), Wherein X1 may be Ile, Val or Met, preferably Ile; X2 may be Tyr, Lys, Arg or Leu, preferably Tyr; X3 may be Ser, Gly or Ala, preferably Ser.
  • the CDR2L sequence is: DXNQRPS (SEQ ID NO 2), where X may be Asn or Thr, preferably Asn.
  • the CDR3L sequence is: QSX1DX2X3LVX4 (SEQ ID NO 3), wherein X1 may be Asn, Ser or Ala, preferably Asn; X2 may be Ala, Met or Leu, preferably Ala; X3 may be Ser, Gly or Ala, preferably Is Ser; X4 may be Glu, Met, Lys, Asp, Thr, Ala or Gly, preferably Glu.
  • CDR1H sequence was: SX1X2MS (SEQ ID NO 4) wherein X1 may be Tyr, Gly, Leu, Val, Met or Ala, preferably Tyr; X2 may be Ala, Ser or Gly, preferably Ala.
  • the CDR2H sequence is: AISSGSGSTXYADSVKG (SEQ ID NO 5), where X can be Tyr, Asn, Leu, Ile, Met or Val, preferably Tyr.
  • the CDR3H sequence is: YX1SX2X3X4SFDY (SEQ ID NO 6), wherein X1 may be Tyr, Trp, Ser, Pro or Ala, preferably Tyr; X2 may be Phe, Leu and Met, preferably Phe; X3 may be Tyr, Val , Pro, Phe or Ala, preferably Tyr; X4 may be Thr, Ala, Gly, or Asn.
  • the present invention provides a human anti-human IFN ⁇ antibody, wherein the light chain comprises the following CDR amino acid sequence, the CDRL1 sequence is set forth in SEQ ID NO. 1, the CDRL2 sequence is set forth in SEQ ID NO. 2, and the CDRL3 sequence is SEQ. ID NO.3 is shown.
  • human anti-human IFN ⁇ antibody provided by the present invention has a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO.
  • the heavy chain variable region thereof comprises the following CDR amino acid sequence, the CDRH1 sequence is shown in SEQ ID NO. 4, the CDRH2 sequence is shown in SEQ ID NO. 5, and the CDRH3 sequence is shown in SEQ ID NO.
  • the human anti-human IFN ⁇ antibody provided by the present invention has a light chain variable region amino acid sequence as shown in SEQ ID NO.
  • the human anti-human IFN ⁇ antibody provided by the present invention has a heavy chain variable region amino acid sequence as shown in SEQ ID NO.
  • the human anti-human IFN ⁇ antibody light chain variable region and heavy chain variable region amino acid sequences provided by the present invention are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively.
  • the antibody provided by the present invention may be one of a single chain antibody, Fab, minibody, chimeric antibody or whole antibody immunoglobulin IgG1, IgG2, IgG4, IgA, IgE, IgM or IgD.
  • the antibodies provided by the invention are whole antibodies or various other forms of genetically engineered antibodies.
  • the anti-IFN ⁇ antibody can be a full antibody or an antibody fragment.
  • the antibody molecule itself can be used for treatment and diagnosis.
  • Antibodies can be labeled, cross-linked or coupled and fused to other proteins or polypeptide molecules to form complexes (such as cytotoxic substances, radionuclides and/or chemical molecules, etc.) for diagnosis and treatment.
  • the present invention further provides an independent gene encoding the antibody, an expression vector, a vector-transfected host cell-related control technique and host cell, an antibody expression sequence, and a method for recovering the antibody in the cell culture supernatant.
  • variable region of the light chain of the above antibody is as shown in 1), 2) or 3) below:
  • the gene encoding the heavy chain variable region of the above antibody is as shown in 1), 2) or 3) below:
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0.1 Ficoll, 42 ° C, etc.; or (3) only having an identity between the two sequences of at least 70% or more Hybridization occurs at least 80% or more, more preferably 90% or more, and more preferably 95% or more.
  • the present invention provides an expression vector comprising the above gene and a host strain, host cell or expression cassette comprising the expression vector.
  • the invention provides a method for preparing a human anti-human IFN- ⁇ antibody, which utilizes a phage display antibody library technology to screen an anti-IFN ⁇ -specific single-chain antibody, obtains an antibody light and heavy chain variable region gene, and clones the same into a whole antibody expression vector. It is fully antibody expressed by a mammalian expression system to obtain its whole antibody protein.
  • nucleotide sequence of the antibody light heavy chain variable region gene is shown in SEQ ID NO. 9 and SEQ ID NO.
  • sequences set forth herein and claimed in SEQ ID NOS. 1-8 include "conservative sequence modifications", i.e., nucleotide and amino acid sequences that do not significantly affect and alter the binding characteristics of the antibody or antibody comprising the amino acid sequence. Modification. Such conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications can be introduced into SEQ ID NOS. 1-8 by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, etc., by performing alanine scanning of the CDR regions of the antibody as in Example 3 of the present invention. Amino acid mutations in some of the sites are examples of conservative sequence modifications.
  • Conservative amino acid substitutions include the replacement of an amino acid residue by an amino acid residue having a similar side chain or other amino acid residue.
  • a family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), with uncharged polar side Chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, guanidine) Acid, leucine, isoleucine, valine, phenylalanine, methionine, amino acids with beta branching side chains (eg threonine, valine, isoleucine) and An amino acid having an aromatic side chain (such as tyrosine, phenylalanine, tryptophan, histidine).
  • an antibody encoded by a nucleotide sequence disclosed herein or/and an antibody comprising an amino acid sequence disclosed herein comprises an antibody that is substantially encoded by a similar sequence modified by a conserved sequence, or an antibody comprising a similar sequence modified by a conserved sequence. All should be considered as the scope of the present invention.
  • the genes encoding the antibodies of SEQ ID NOS. 1 to 8 of the present invention may be, but are not limited to, SEQ ID NOS. 9 and 10, for example, in the coding region thereof, without changing the amino acid.
  • the gene sequence encoding the above antibody is modified to obtain a gene encoding the same antibody.
  • One skilled in the art can artificially engineer the gene according to the codon preference of the expression antibody host to increase the expression efficiency of the antibody.
  • the invention also provides an antibody-targeting drug molecule comprising the above-described human anti-human IFN ⁇ antibody linked to a chemical molecule, a radioisotope, a polypeptide molecule, a toxin or a biomacromolecule.
  • the means of attachment is that the antibody is labeled, in vivo, or molecularly coupled.
  • the present invention also provides a bispecific or multispecific molecule comprising the antigen binding site of the above human anti-human IFN ⁇ antibody or antibody.
  • a fusion protein of an antibody with other proteins or/and polypeptides comprising a complex of the above human anti-human IFN ⁇ antibody and a functional protein or polypeptide molecule.
  • the above fusion protein is a recombinant expression vector obtained by ligating an antibody gene and a fusion protein gene, and obtaining a recombinant fusion protein molecule through a mammalian cell or other expression system.
  • the present invention also provides a medicament, preparation or detection reagent containing the above human anti-human IFN ⁇ antibody.
  • the invention also provides an antibody-containing component and a pharmacologically acceptable delivery molecule or solution.
  • the therapeutic component is sterile and can be lyophilized at low temperature.
  • the present invention provides the use of a human anti-human IFN ⁇ antibody for the preparation of a therapeutic agent for diseases in which IFN ⁇ is targeted.
  • the disease is systemic lupus erythematosus, insulin-dependent diabetes mellitus, autoimmune thyroiditis, psoriasis, polymyositis, dermatomyositis, sclerosis, rheumatoid arthritis and the like.
  • the present invention provides an antibody against multiple subtypes of human IFN[alpha] which inhibits one or more of the biological activities exhibited by IFN[alpha].
  • This antibody acts by blocking the binding of IFN ⁇ to its receptor, and can also act by reducing the level of IFN ⁇ in the body. All interfering functions possessed by IFN ⁇ antagonists should be considered equally for the purposes of the present invention.
  • Applicants utilize a self-constructed library capacity of 1.35 ⁇ 10 10 large-capacity fully synthetic phage single-chain human antibody library (ZL.200910091261.8), with IFN ⁇ _1b and IFN ⁇ _2b as targets, and obtained by alternate screening.
  • a functional antibody AIA22 against a plurality of subtypes of human IFN ⁇ , the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the amino acid sequence of the AIA22 antibody And DNA sequences are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.
  • the present invention uses a phage display technology to screen a single-stranded genetically engineered antibody specific for human IFN ⁇ by multiple rounds of bio-panning from a large-capacity fully synthetic human single-chain antibody library (single chain) Variable fragment, scFv).
  • the above-mentioned antibody light heavy chain variable region genes are separately cloned into the whole antibody mammalian cell transient expression vectors pABL and pABG1, and transiently secreted and expressed by co-transfection of HEK293-T cells, and affinity purification is obtained.
  • Antibody AIA22 is separately cloned into the whole antibody mammalian cell transient expression vectors pABL and pABG1, and transiently secreted and expressed by co-transfection of HEK293-T cells, and affinity purification is obtained.
  • Antibody AIA22 is separately cloned into the whole antibody mammalian cell transient expression vectors pABL and p
  • the antibody AIA22 described in the present invention has a good therapeutic application prospect, mainly manifested by Specific binding activities of multiple subtypes such as IFN ⁇ -1, IFN ⁇ -2, IFN ⁇ -4, IFN ⁇ -5, IFN ⁇ -6, IFN ⁇ -8, IFN ⁇ -14, IFN ⁇ -17, and IFN ⁇ -21 can effectively block It inhibits the proliferation of Daudi cells.
  • the result showed that the affinity of the antibody Fab K D IFN ⁇ -2b is 0.213nM, a fusion protein with HSA-IFN ⁇ _1b affinity K D of 3.24 nM.
  • the present invention screens a full-human antibody against multiple subtypes of human IFN ⁇ and a series of mutant antibodies whose affinity remains unchanged or increased by large-capacity synthetic antibody library technology, and confirms that the antibodies of the invention are specific to IFN ⁇ . Combine and inhibit its biological function.
  • the fully human anti-IFN ⁇ genetically engineered antibody variable region gene obtained above and the whole antibody gene under the characteristics of the antibody sequence, the antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system, or Based on this modified other antibody gene containing this antibody gene, an antibody product having an activity of inhibiting IFN ⁇ or a complex obtained by in vitro labeling or cross-linking is obtained, and is clinically used for treatment and IFN ⁇ .
  • Specific antibody drugs for related autoimmune diseases are provided.
  • Figure 1 shows the vector map, wherein Figure 1A is the pABL vector map, Figure 1B is the pABK vector map, and Figure 1C is the pABG1 vector map.
  • FIG. 2 is expressed using FreeStyle TM HEK293-T and transient expression system AIA22, Sifalimumab whole antibodies native SDS-PAGE electrophoresis pattern obtained by ProteinA affinity purification columns; 1: Pr Marker, 2: AIA22,3: Sifalimuamb.
  • Figure 3 shows the results of ELISA-specific identification of AIA22 whole antibodies in combination with multiple types of antigens in a concentration gradient; the legend shows the antigen type and its coating concentration.
  • Figure 4 shows the affinity curves of AIA22 with IFNa-2b and HSA-IFNa-1b using the BIAcore 3000 system.
  • 4A In order to determine the affinity of AIA22 and IFN ⁇ -2b, the concentration of IFN ⁇ -2b represented by the top to bottom curve was 20nM, 10nM, 5nM, 2.5nM, 1.25nM and 0.625nM, respectively;
  • FIG. 4B for determining AIA22 and As a result of HSA-IFN ⁇ -1b affinity, the HSA-IFN ⁇ -1b concentration represented by the top to bottom curve was 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.
  • Figure 5 shows the results of AIA22 and Sifalimuab blocking the inhibition of proliferation of Daudi cells by IFN ⁇ _2b; the IFN ⁇ _2b used inhibited the proliferation of Daudi at a concentration of 25 pM.
  • Figure 6 is a graph showing the neutralization activity of AIA22 against different IFN ⁇ subtypes.
  • the ordinate indicates the ratio of the EC50 value of AIA22 to different subtypes and the concentration of each subtype of IFN ⁇ . The smaller the ratio, the stronger the neutralizing activity of AIA22 on this subtype.
  • Figure 7 shows the results of the inhibition of proliferation of different subtypes of IFN ⁇ by AIA22 partial mutants
  • Figure 7A IFN ⁇ _2b was used to block IFN ⁇ _2b activity at a concentration of 25 pM
  • Figure 7B Blocking IFN ⁇ _1a activity, IFN ⁇ _1a The concentration used was 36 pM.
  • the large-capacity fully synthetic phage single-chain antibody library was constructed by the Chinese Academy of Military Medical Sciences (ZL200910091261.8) with a storage capacity of 1.35 ⁇ 10 10 .
  • the antigens for screening the antibody library were human IFN ⁇ _2b (Israel ProSpec-Tany) recombinantly expressed in E. coli and HSA-IFN ⁇ _1b and HSA-IFN ⁇ _2b (antibody online company) fused to human serum albumin (HSA).
  • HSA human serum albumin
  • the strain was XL1-Blue (Stratagene, USA); the phage used was M13KO7 (Invitrogene, USA).
  • the variable region nucleic acid sequence synthesis service of MedImmune's Sifalimumab is provided by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.
  • the frozen antibody library was taken and cultured in a medium of 200 ml 2 ⁇ YT-CTG (the final concentration of chloramphenicol C was 100 ug/mL, the final concentration of tetracycline T was 10 ug/mL, and the final concentration of Glucose was 0.5%) at 37 ° C, 220 rpm.
  • the IFN ⁇ _2b and IFN ⁇ 1b were used as antigens for alternate screening: the recombinant protein antigen IFN ⁇ _2b was diluted with a coating solution to 20 ⁇ g/ml (the second and third rounds were 5 ug/mL and 1 ug/mL, respectively), and the tube was coated with 1 ml/tube. , 4 ° C coated overnight. On the next day, the immunotubes were washed twice with PBS for 2 min/pass, and then blocked with 2% BSA at 37 °C for 2 h (after repeated use of 0.2% casein and 2% BSA in each subsequent round).
  • the prepared phage antibody library was blocked with a blocking solution (with immunotube blocking solution, 0.1% Tween 20, coated with antigen as HSA fusion protein, and correspondingly added to HSA at a final concentration of 40 ug/mL) for 30 min at 37 °C.
  • the pre-blocked phage antibody library was added to the blocked immunotube and allowed to act overnight at 4 °C.
  • the solution in the immunotube was discarded, and after washing with PBST, 0.2 mol/L of Glycine-HCl (pH 2.2) was eluted, and 1 mol/LTris was neutralized to pH 7.4.
  • the eluate was infected with E. coli XL1-Blue in logarithmic growth phase, coated on 2 ⁇ YT-CTG plates, and cultured overnight at 37 °C. The overnight bacteria were subjected to phage display. For the next round of screening.
  • HSA-IFN ⁇ _1b and HSA-IFN ⁇ _2b were each diluted to 2 ⁇ g/ml with a coating solution, and added to a 96-well enzyme-linked plate, 50 ⁇ l/well, and coated at 4 ° C overnight.
  • the pre-blocked mouse anti-M13 antibody was added to the enzyme-linked plate, 50 ⁇ l/well, and allowed to stand at 37 ° C for 30 min. The liquid was discarded, and the enzyme-linked plate was washed twice with PBST and once with PBS, 200 ⁇ l/well for 5 min each time. Discard the washing solution, add OPD substrate coloring solution, 50 ⁇ l/well, and let stand to develop color at room temperature. The color development was stopped with 2M H 2 SO 4 . The absorbance value OD492/630 was measured by a microplate reader.
  • Coating buffer (pH 9.6): 1.59 g of Na 2 CO 3 , 2.93 g of NaHCO 3 , and double distilled water to 1 L;
  • Blocking solution 1 ⁇ PBS + 2% BSA (or 0.2% casein) + 0.1% Tween20;
  • Washing solution PBST 1 ⁇ PBS + 0.1% Tween20;
  • OPD substrate dilution 0.2 M Na 2 HPO 4 (28.4 g/L) 25.7 ml, 0.1 M citric acid (19.2 g/L) 24.3 ml, distilled water, 50 ml.
  • the V L1 variable region gene was cloned into the vector PABL using the restriction sites BsrG I and Hind III, and the V H3 variable region gene was cloned into the vector pABG1 using the restriction sites Afl II and NheI.
  • the recombinant plasmid was transformed into E.
  • coli Top10 and the recombinant plasmid was identified by bacterial PCR and sequencing to obtain the correct expression vector of the whole antibody light and heavy chain. Large plasmid will provide the antibody light and heavy chains in a molar ratio of 1: 1 transfection of FreeStyle TM HEK293-T cells, transient expression of full antibodies, affinity chromatography expression supernatant was purified by ProteinA, and the whole purified Antibodies are subjected to electrophoretic analysis, affinity and specificity assays, and the like.
  • the amino acid sequence of the Sifalimumab light heavy chain is SEQ ID NO. 19 and SEQ ID NO.
  • the variable region nucleotide sequence is designed as SEQ ID NO. 17 and SEQ ID NO. 18, and the restriction sites Xba I and Kas I are introduced at both ends of the light chain, and the restriction site Afl II is introduced at both ends of the heavy chain.
  • Nhe I cloned into vectors pABK and pABG1, respectively, by restriction enzyme ligation. The recombinant plasmid was transformed into E.
  • the recombinant plasmid was identified by bacterial PCR and sequencing to obtain the correct expression vector of the whole antibody light and heavy chain. After the plasmid was extracted, the antibody light and heavy chain were transfected into HEK293-T cells at a molar ratio of 1:1, and the transient expression of the whole antibody was performed. The expression supernatant was purified by Protein A affinity chromatography column, and the subsequent antibody was used as a control antibody. .
  • the monoclonal antibody obtained by the third round of IFN ⁇ _2b screening was identified, and the specific phage antibody was named as AIA22, and the phage single-chain antibody specifically binds to IFN ⁇ _2b and INF ⁇ _1b, and the amino acid and nucleotide sequence of the single-chain antibody is SEQ ID NO. As shown in SEQ ID NO. 7 and SEQ ID NO. 8, the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in .15 and SEQ ID NO.
  • the light and heavy chain variable region genes of phage antibody AIA22 and the light and heavy chain genes of Sifalimumab were cloned into the whole antibody transient expression vectors pABL and pABG1, respectively, and the whole antibody light and heavy chain recombinant expression vector was constructed and passed through HEK293-
  • the transient expression system of T cells achieves transient secretion expression of mammalian cells. After purification by Protein A column, the whole antibody protein was obtained.
  • the results of SDS-PAGE electrophoresis are shown in Figure 2. The antibody purity is high, and the electrophoresis band position is in line with expectations, which can be used in subsequent experiments.
  • AIA22 Specificity of AIA22 was analyzed at the full antibody level, with HSA-IFN ⁇ _1b and HSA-IFN ⁇ _2b as positive antigens, IFN ⁇ , HSA, His-IL6R (His-tagged IL6R), VEGF, TNF, AHC-A ⁇ -T (AHC, A ⁇ fragment and botulinum toxin fusion protein) and HEK293 fragmented cell supernatant were used as control antigens, coated with enzyme-linked plates at 4 °C overnight, and purified by gradient dilution of AIA22 whole antibody, with HRP-labeled anti-human IgG antibody. The anti-ELISA was performed to detect the specific binding activity of the AIA22 whole antibody. As a result, as shown in Fig. 3, it can be seen that the binding of AIA22 to the target antigens HSA-IFN ⁇ _1b and HSA-IFN ⁇ _2b is specific.
  • CM5 chip was purchased from GE Healthcare Life Sciences. Subtypes of IFN ⁇ _1, IFN ⁇ _2, IFN ⁇ _4, IFN ⁇ _5, IFN ⁇ _6, IFN ⁇ _8, IFN ⁇ _10, IFN ⁇ _14, IFN ⁇ _16, IFN ⁇ _17, and IFN ⁇ _21 were purchased from PBL (Cat. No. 11002-1). Daudi cells (human Burkitt's lymphoma cells) were purchased from the Cell Bank of the Institute of Culture Collection of the Chinese Academy of Sciences. CCK-8 was purchased from Japan Tongren Chemical Co., Ltd. Newborn bovine serum NCS was purchased from PAA. RPMI 1640 cell culture medium was purchased from Gibco. Other materials involved are the same as in Example 1.
  • the purified antibody AIA22 was diluted to 1 ug/mL with HBS-EP buffer and coupled to a CM5 chip pre-coated with Prot-G at a temperature of 25 ° C, a flow rate of 5 ⁇ L/min, and a coupling target value. It is 200RU.
  • the antigen IFN ⁇ _2b was used as a mobile phase and diluted with a two-fold gradient with HBS-EP at a concentration ranging from 20 nM to 625 pM. Test conditions: temperature 25 ° C, flow rate 30 ⁇ L / min; binding time 3 min, dissociation time 15 min.
  • the chip was regenerated, and the regeneration conditions were: 3M MgCl 2 , 30 uL/min ⁇ 30 s, and after the regeneration, AIA22 of the same target value was continuously coupled, and the next reaction was carried out.
  • the blank control and irrelevant antibody control response values were subtracted, and the results were analyzed by BIAevaluation software.
  • IFN ⁇ The function of IFN ⁇ , including its role in the pathology of systemic lupus erythematosus (SLE), is achieved by binding to the cell surface type I interferon receptor IFNAR and activating the downstream JAK-STAT pathway. The same is true for IFN ⁇ to inhibit proliferation of Daudi cells. Human IFN ⁇ inhibited the proliferation of Daudi cells cultured in vitro, and there was a certain dose-effect relationship. By evaluating the binding of AIA22 to the binding of human IFN ⁇ to IFNAR in vitro and promoting the proliferation of Daudi cells, it can indirectly reflect its actual role in the treatment of diseases.
  • the cytotoxicity curves of different subtypes of human IFN ⁇ inhibiting Daudi proliferation were investigated by inhibition experiments to determine the effective inhibitory concentration; on the basis of this, the in vitro neutralizing activity of anti-human IFN ⁇ antibody AIA22 was verified by antibody blocking assay.
  • the specific operations are as follows:
  • each interferon The type occupies 2 columns, corresponding to 14 concentration gradients of each antibody.
  • each concentration of the antibody corresponds to a control containing no interferon in one well, that is, 8 ⁇ 4 wells are required for each pair of mAb/IFN, and 2 duplicate wells are set at the same time.
  • the total number of cell wells to be inoculated is calculated as follows);
  • the BIAcore 3000 system measures antibody affinity:
  • the CM5 chip was coupled with the antibody AIA22, and the concentration gradients of HSA-IFN ⁇ _1b and IFN ⁇ _2b were used as mobile phases.
  • the affinity of the antibody to HSA-IFN ⁇ _1b and IFN ⁇ _2b was determined by BIAcore 3000 system.
  • the curve characteristics were analyzed by BIAevaluation software. The appropriate fitting interval was selected, the curve was fitted and the antibody affinity was calculated (Fig. 4A and Fig. 4B). The results are shown in the following table.
  • the Daudi cell surface expresses an IFNAR receptor, and IFN ⁇ inhibits the proliferation of the cell by binding to the INFAR receptor.
  • the binding of the antibody to IFN ⁇ can block the signal transduction between IFN ⁇ and the receptor, thereby blocking the inhibition of cell proliferation by IFN ⁇ , and recovering the proliferative activity of the cell. Blocking efficiency is manifested by cellular activity.
  • the present invention uses the Daudi proliferation assay to detect the neutralizing activity of AIA22 on various subtypes of human IFN ⁇ , and calculates the EC 50 value by fitting. The results are shown in Fig. 5, Fig. 6 and Table 2. Among them, the results of evaluation of the in vitro activity of AIA22 and Sifalimumab against IFN ⁇ _2b are shown in Fig. 5.
  • the "1000 x" column indicates the neutralizing activity when the antibody concentration is 1000 times the IFN? concentration.
  • the site-directed mutagenesis primers were designed to perform alanine scanning on the six CDR regions of the AIA22 antibody, and if they were alanine at this position, they were mutated to Gly and Ser, respectively.
  • the method was carried out by the method of site-directed mutagenesis.
  • the specific method can be referred to the literature [Wang Ronghao, Chen Ruichuan, Liu Runzhong. An optimization method for rapid point mutation. Journal of Xiamen University (Natural Science Edition), 2008, Vol 47, sup 2, 282-285].
  • the antigens (HSA-IFN ⁇ _1b and HSA-IFN ⁇ _2b) were each diluted to 3 ⁇ g/ml with a coating solution, and added to a 96-well enzyme-linked plate, 50 ⁇ l/well, and coated at 4 ° C overnight. The next day, the solution was discarded, and the enzyme-linked plates were blocked with PBS containing 2% skim milk powder, 50 ⁇ l/well, 37 ° C, 30 min. The blocking solution was discarded, and parental antibodies and mutant antibodies (initial concentration 1 ug/mL) diluted in PBST milk (2% skim milk powder, 0.1% Tween 20) were added, 50 ⁇ l/well, and 37 ° C, and allowed to stand for 1 h.
  • PBST milk 2% skim milk powder, 0.1% Tween 20
  • the antibody to be detected was diluted to 100 nM with HEPES-EP buffer, coated in Anti-Human IgG Fc Capture, coating time was 20 min; after the coating was completed, it was washed with HEPES-EP buffer for 2 min, and after baseline calibration for 1 min, Anti- Human IgG Fc Capture was transferred into IFN ⁇ samples diluted to 20 nM with HEPES-EP buffer for 10 min. After the binding curve reached equilibrium, Anti-Human IgG Fc Capture was transferred to a new HEPES-EP buffer system for antigen-antibody complex solution. The dissociation time is 15 min. After the test is completed, Affinity was analyzed using the accompanying Data Analysis analysis software.
  • the specific method is the same as the 2.2.2 anti-IFN ⁇ antibody AIA22 in vitro.
  • the basic principle of antibody dosage is to refer to the previous experimental data, and take appropriate concentration gradient samples on both sides of the corresponding concentration.
  • mutations at multiple sites can ensure the affinity of the antibody for IFN ⁇ -2b.
  • the affinity for IFN ⁇ _1b was significantly increased without significant changes; at the same time, there was a certain improvement in blocking the antiproliferative activity of IFN ⁇ _1b, and the results of cytological activity are shown in Fig. 7A and Fig. 7B.
  • the corresponding value in the table relative to the AIA22 affinity column is the ratio of the dissociation constant Kd of the mutant antibody calculated to the Kd of the parent antibody. The smaller the ratio, the more significant the affinity is increased; on the contrary, the larger the value, the lower the affinity. obvious.
  • the fully human antibody against multiple subtypes of human IFN ⁇ of the present invention and a series of mutant antibodies whose affinity remains unchanged or increased specifically bind to IFN ⁇ and inhibit its biological function Using the fully human anti-IFN ⁇ genetically engineered antibody variable region gene obtained by the present invention and the whole antibody gene under the characteristics of the antibody sequence, the antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system, or Based on this Any other gene having such an antibody gene, which obtains an antibody product having a biological activity of inhibiting IFN ⁇ , or a complex obtained by an in vitro labeling or cross-linking method, is clinically used for the treatment of an autoimmune disease associated with IFN ⁇ . Specific antibody drugs.

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Abstract

L'invention concerne un anticorps humain anti-interféron alpha (IFNα) humain anticorps et un gène codant et son application. Par combinaison d'un procédé de génie génétique avec une technologie d'affichage à la surface de bactériophage, une pluralité d'anticorps à chaîne de signalisation, obtenus par ingénierie génique, anti-sous-type d'IFNα humain, est criblée à partir d'une bibliothèque d'anticorps humains à chaîne unique entièrement synthétique et une séquence génique d'une région variable des anticorps est obtenue ; des supports recombinants d'anticorps holistiques monoclonaux Fab et IgG sont construits sur la base ci-dessus et une molécule d'anticorps de haute pureté est obtenue par l'intermédiaire de l'expression et de la purification de cellules mammifères. L'affinité de la combinaison de l'anticorps Fab obtenu et d'IFNα-2 humain n'est pas supérieure à 2 nM et l'affinité de la combinaison de l'anticorps Fab obtenu et d'IFNα-1 n'est pas supérieure à 100 nM. L'anticorps de la présente invention inhibe efficacement l'activité biologique d'une pluralité de sous-types d'IFNα, tels que IFNα-2 au niveau cellulaire et la présente invention concerne un médicament de type anticorps spécifique pour le traitement de maladies associées à l'interféron, telles que le lupus érythémateux disséminé et d'autres maladies auto-immunes.
PCT/CN2015/070625 2015-01-13 2015-01-13 Anticorps humain anti-interféron alpha humain et son application WO2016112497A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113521276A (zh) * 2021-07-13 2021-10-22 江苏荃信生物医药有限公司 包含抗人干扰素α受体1（IFNAR1）单克隆抗体的液体制剂
WO2022155541A1 (fr) 2021-01-14 2022-07-21 AskGene Pharma, Inc. Promédicaments d'interféron et leurs procédés de fabrication et d'utilisation

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CN1492930A (zh) * 2001-02-22 2004-04-28 �����ɷ� 抗-干扰素α的抗体
CN102344491A (zh) * 2003-12-10 2012-02-08 米德列斯公司 干扰素α抗体及其用途

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Publication number Priority date Publication date Assignee Title
CN1492930A (zh) * 2001-02-22 2004-04-28 �����ɷ� 抗-干扰素α的抗体
CN102344491A (zh) * 2003-12-10 2012-02-08 米德列斯公司 干扰素α抗体及其用途

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WU, MEIYING ET AL.: "Preparation and application of monoclonal antibodies to recombinant human IFN-a", CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY, vol. 16, no. 3, 30 September 2002 (2002-09-30) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022155541A1 (fr) 2021-01-14 2022-07-21 AskGene Pharma, Inc. Promédicaments d'interféron et leurs procédés de fabrication et d'utilisation
CN113521276A (zh) * 2021-07-13 2021-10-22 江苏荃信生物医药有限公司 包含抗人干扰素α受体1（IFNAR1）单克隆抗体的液体制剂

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