WO2018006824A1 - 一种BLyS抗体及其制备方法和应用 - Google Patents

一种BLyS抗体及其制备方法和应用 Download PDF

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WO2018006824A1
WO2018006824A1 PCT/CN2017/091839 CN2017091839W WO2018006824A1 WO 2018006824 A1 WO2018006824 A1 WO 2018006824A1 CN 2017091839 W CN2017091839 W CN 2017091839W WO 2018006824 A1 WO2018006824 A1 WO 2018006824A1
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seq
sequence
amino acid
variable region
sequence listing
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PCT/CN2017/091839
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French (fr)
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曹晓丹
胡颖莹
任芳
宫世勇
公静
吕强
顾红专
施蓓蕾
邵小慧
吕晓芬
梁绍勤
刘礼乐
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上海开拓者生物医药有限公司
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Priority to US16/315,519 priority Critical patent/US20210347873A1/en
Priority to EP17823636.0A priority patent/EP3483181A4/en
Priority to CN201780033470.XA priority patent/CN109311988A/zh
Priority to JP2018562628A priority patent/JP2019533423A/ja
Publication of WO2018006824A1 publication Critical patent/WO2018006824A1/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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of antibodies, and in particular relates to a BLyS antibody and a preparation method and application thereof.
  • Autoimmune disease is a disease in which the body's own immune system attacks its normal organs, tissues and cells. It is a chronic disease that becomes weak when it develops into the body and cannot be cured. It can only be controlled. This results in patients being burdened with high medical costs and a sharp decline in the quality of life, placing a heavy burden on patients and their families and even society.
  • the autoimmune diseases familiar to the public include systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). SLE may affect various organs, and it is currently impossible to predict when it will occur. The natural course of the disease is often manifested by aggravation and remission of the disease.
  • SLE The global average prevalence of SLE is 12-39 per 100,000 people, and the prevalence rate in China is 30-70 per 100,000 people, second only to blacks (100/100,000), ranking globally.
  • SLE usually occurs in young women, and 90% of patients are women.
  • RA is a long-term persistent disease that mainly affects joints. It usually causes joint fever, swelling and pain. In severe cases, it can cause erosion and damage of the joint surface and even cause limb malformation. According to statistics, the incidence of rheumatoid arthritis is about 0.3%. In recent years, the incidence of autoimmune diseases in China and the world has gradually increased. Due to China's vast territory and large population, the number of patients calculated according to this incidence rate is quite large. However, there is still a lack of interventional treatments that are very effective, have few side effects, and are highly specific to early block the damage of target organs and improve the prognosis of patients.
  • B lymphocyte stimulator can be detected in patients with autoimmune diseases and B cell tumors.
  • serum levels of BLyS are found to increase in multiple groups of patients with systemic lupus erythematosus (SLE), and this correlates with autoimmune antibody production and disease activity index [see Stohl et al. 2003, Arthritis Rheum, 48). (12): 3475; Petri et al. 2008, Arthritis Rheum, 58(8): 2453].
  • High levels of BLyS were also found in the synovial fluid of patients with rheumatoid arthritis (RA) [see Tan et al. 2003, Arthritis Rheum, 48(4): 982].
  • RA rheumatoid arthritis
  • BLyS also known as BAFF, THANK, TALL-1, TNFSF13B or zTNF4, is a member of the tumor necrosis factor (TNF) ligand superfamily [see Baker et al. 2003, Arthritis Rheum, 48(11): 3253] .
  • BLyS is a type II transmembrane protein consisting of 285 amino acids with both membrane-bound and 152 amino acid soluble forms after cleavage. BLyS is expressed on monocytes, macrophages and dendritic cells and is up-regulated under stimulation with interferon gamma and interleukin-10.
  • recombinant human BLyS can enhance B cell proliferation and antibody secretion by binding to major receptors on the surface of B cells.
  • recombinant human BLyS causes spleen hyperplasia in mice, which is mainly due to an increase in the number of mature B cells. Injection of BLyS into mice also results in an increase in the concentration of antibodies in the serum, as well as an increase in T cell-dependent and non-antigen-dependent humoral immunity. Overexpression of BLyS produces antibodies with abnormally high levels of expression, leading to SLE, RA and other autoimmune diseases.
  • the technical problem to be solved by the present invention is to provide a humanized or fully humanized BLyS antibody with high affinity and specificity, and a preparation method and application thereof, in order to overcome the shortcomings of the current lack of effective and safe BLyS antibodies.
  • the BLyS antibody of the present invention has high affinity with BLyS; inhibits binding of BLyS to its receptor; inhibits proliferation of mouse B cells induced by human BLyS; and lacks cross-reactivity with BLyS homologous protein antigen such as human APRIL, Therefore, it can be used for the preparation of a medicament for treating or preventing a disease associated with abnormal expression or dysfunction of BLyS such as an autoimmune disease or a tumor.
  • the present inventors used a phage display and hybridoma technique to obtain a lead antibody of a BLyS antibody.
  • the antibody has high affinity (affinity KD ⁇ 5 ⁇ 10 -9 M), can effectively block the binding of BLyS to the receptor, and lack cross-reactivity with the same protein antigens such as human ARRIL.
  • the amino acid sequence of the heavy chain variable region of the BLyS antibody and the light chain variable region of the BLyS antibody is then sequenced by molecular biological methods.
  • the invention provides an isolated protein comprising the complementarity determining regions (CDRs or CDRs) of a BLyS antibody: one or more of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and/or a light of a BLyS antibody
  • CDRs or CDRs complementarity determining regions
  • One or more of the chain CDR1, the light chain CDR2 and the light chain CDR3, the amino acid sequence of the heavy chain CDR1 is SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID in the sequence listing No. 26, SEQ ID No. 34, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35 SEQ ID No. 43, SEQ ID No. 51, SEQ ID No. 59, SEQ ID No. 67, SEQ ID No. 75, SEQ ID No. 83, SEQ ID No. 91 or SEQ ID No. 99
  • the amino acid sequence of the heavy chain CDR3 is SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44, SEQ in the Sequence Listing. ID No. 52, SEQ ID No.
  • amino acid sequence of the light chain CDR1 is SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 22 in the sequence listing, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46, SEQ ID No. 54, SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 86, SEQ ID No. 94 or SEQ ID No. 102;
  • amino acid sequence of the light chain CDR2 is SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No.
  • SEQ ID No. 39 SEQ ID No. 47, SEQ ID No. 55, SEQ ID No. 63, SEQ ID No. 71, SEQ ID No. 79, SEQ ID No. 87, SEQ ID No. 95 or SEQ ID No. 103.
  • the amino acid sequence of the light chain CDR3 is SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. in the sequence listing. 48, SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96 or SEQ ID No. 104;
  • the amino acid sequence of the heavy chain CDR1 is as SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42 in the sequence listing.
  • the amino acid sequence represented by SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 90 or SEQ ID No. 98 has at least 80 The amino acid sequence of % sequence homology is shown; the amino acid sequence of the heavy chain CDR2 is as SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ in the sequence listing. ID No. 35, SEQ ID No.
  • amino acid sequence represented by ID No. 99 is represented by an amino acid sequence having at least 80% sequence homology; the amino acid sequence of the heavy chain CDR3 is SEQ ID No. 4, SEQ ID No. 12, SEQ in the sequence listing. ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44, SEQ ID No. 52, SEQ ID No. 60, SEQ ID No. 68, SEQ ID No. 76, SEQ ID No. .84, SEQ ID No. 92 or SEQ ID
  • amino acid sequence of the light chain CDR1 is as SEQ ID No. 6, SEQ ID No. 14, SEQ ID in the sequence listing. No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46, SEQ ID No. 54, SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 86.
  • the amino acid sequence represented by SEQ ID No. 94 or SEQ ID No. 102 has an amino acid sequence of at least 80% sequence homology; the amino acid sequence of the light chain CDR2 is SEQ ID No. in the sequence listing. 7. SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No.
  • SEQ ID No. 47 SEQ ID No. 55, SEQ ID No. 63, SEQ ID No. 71, An amino acid sequence having at least 80% sequence homology of the amino acid sequence represented by SEQ ID No. 79, SEQ ID No. 87, SEQ ID No. 95 or SEQ ID No. 103; amino acid of the light chain CDR3
  • the sequence is as SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. 48, SEQ ID No. 56, SEQ ID in the sequence listing. No.64, SEQ ID No. 72, S The amino acid sequence of EQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96 or SEQ ID No. 104 has an amino acid sequence of at least 80% sequence homology.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 The sequence is shown in SEQ ID No. 4 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 11 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the sequence listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is as in the sequence listing.
  • SEQ ID No. 19 and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the Sequence Listing;
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 26 of the Sequence Listing,
  • the amino acid sequence of the CDR2 of the chain is shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 28 of the Sequence Listing;
  • the amino acid sequence of the CDR1 of the heavy chain is SEQ ID No.
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No.
  • amino acid sequence of CDR3 is shown in SEQ ID No. 36 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is SEQ ID No. 43 of the Sequence Listing. As shown, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 44 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 50 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 As shown in the Sequence Listing SEQ ID No. 51, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 58 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 59 of the sequence listing
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 60 of the sequence listing
  • the amino acid sequence of the heavy chain CDR1 is as in the sequence listing.
  • SEQ ID No. 66 the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 67 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 68 of the Sequence Listing
  • Chain CDR1 The amino acid sequence is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 75 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 76 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 82 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 83 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 is as
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 90 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 91 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 92 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 98 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is SEQ ID of the Sequence Listing. And the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 100 of the Sequence Listing;
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 7 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is as in the Sequence Listing.
  • SEQ ID No. 8 the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 14 of the Sequence Listing, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 15 of the Sequence Listing, and the light
  • the amino acid sequence of the CDR3 of the chain is shown in SEQ ID No. 16 of the Sequence Listing; the amino acid sequence of the CDR1 of the light chain is shown in SEQ ID No.
  • amino acid sequence of the CDR2 of the light chain is SEQ ID No. of the Sequence Listing.
  • amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 24 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 30 of the Sequence Listing, the amino acid of the light chain CDR2 The sequence is shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 32 of the Sequence Listing; the amino acid sequence of the CDR1 of the light chain is shown in SEQ ID No. 38 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No.
  • the acid sequence is shown in SEQ ID No. 40 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 46 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 47 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 48 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is as The sequence of the SEQ ID No. 55, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 62 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 63 of the sequence listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 64 of the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in the sequence listing.
  • ID No. 70 the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 71 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 72 of the Sequence Listing;
  • the amino acid sequence of CDR1 is shown in SEQ ID No.
  • amino acid sequence of the light chain CDR2 is as a sequence.
  • SEQ ID No. 79, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 80 of the sequence listing;
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 86 of the sequence listing, the light
  • the amino acid sequence of the chain CDR2 is shown in the sequence listing SEQ. ID No. 87, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 88 of the Sequence Listing;
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 94 of the Sequence Listing, the light chain
  • the amino acid sequence of CDR2 is shown in SEQ ID No.
  • the above protein comprises a heavy chain variable region of a BLyS antibody consisting of the above CDR region and a framework region and/or a light chain variable region of a BLyS antibody, the amino acid sequence of the heavy chain variable region being in the sequence listing SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 57, SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 89 or SEQ ID No. 97; the amino acid sequence of the light chain variable region is SEQ ID No. 5 in the sequence listing, SEQ ID No.
  • the present invention also provides an isolated protein comprising a heavy chain variable region of a BLyS antibody and/or a light chain variable region of a BLyS antibody, the amino acid sequence of the heavy chain variable region being SEQ ID No. in the Sequence Listing. 1. SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 57, SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 89 or SEQ ID No. 97; the amino acid sequence of the light chain variable region is SEQ ID No. 5, SEQ ID No. in the sequence listing. 13. SEQ ID No. 21, SEQ ID No.
  • SEQ ID No. 37 29, SEQ ID No. 37, SEQ ID No. 45, SEQ ID No. 53, SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 85, SEQ ID No. 93 or SEQ ID No. 101.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the variable region of the chain is shown in SEQ ID No. 9 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 13 of the Sequence Listing;
  • the amino acid sequence of the variable region of the heavy chain The sequence of the light chain variable region is shown in SEQ ID No. 21;
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing, and The amino acid sequence of the light chain variable region is set forth in SEQ ID No. 37 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 41 of the Sequence Listing, and the amino acid of the light chain variable region The sequence is shown in SEQ ID No. 45 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing, and The amino acid sequence of the variable region of the chain is shown in SEQ ID No. 53 of the Sequence Listing; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID No.
  • the binding regions of the antibody and the antigen each comprise a light chain variable region and a heavy chain variable region, each variable region comprising three domains, CDR1, CDR2 and CDR3.
  • the number in Table 1 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of 2-1G11 is SEQ ID No. 1, and the CDR1 of the heavy chain protein variable region of 2-1G11 The amino acid sequence is shown in SEQ ID No. 2.
  • the isolated protein of the present invention may further comprise a framework region (or framework region or framework region) of the BLyS antibody,
  • the framework region comprises a heavy chain framework region and/or a light chain framework region; preferably, the heavy chain framework region is a human or murine antibody heavy chain framework region, and/or the light chain framework region is a human or murine antibody Light chain framework area.
  • the heavy chain framework region is a human antibody heavy chain framework region
  • the human antibody heavy chain framework region residue may comprise germline DP4, DP7, DP8, DP9, DP10, DP14 (V H 1-18), DP31, DP33, DP35 (V H 3-11), DP45, DP46, DP47, DP48, DP49 (V H 3-30), DP50, DP51 (V H 3-48), DP53, DP54 (V H 3-7), DP65, DP66, DP67, DP68 and DP69, especially FR1, FR2, FR3 of these germ lines; and JH fragment J H -1, J H - 2.
  • the light chain framework region is a human antibody light chain framework region, and the human antibody light chain framework region residues may comprise germline O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26 (A10) and DPK 28, in particular FR1, FR2 of these species FR3; and sequences encoded by Jk fragments Jk1, Jk2, Jk3, Jk4 and JK5, in particular FR4 of these lines.
  • Such framework region sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (http://www2.mrc-lmb.cam.ac.uk/vbase/), as well as Found in Kabat (EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th Edition).
  • the outer region heavy chain framework residues preferably human germline antibody heavy chain V H exon V H 1-18, the outer V H exon in the present invention, a humanized antibody of the preferred embodiment of 3- 7 or the outer exon J H J H -6, light chain framework region residues preferably human germline antibody light chain exon V K B3, an outer J K exons or V K J K -4 significant A10 of the child.
  • the protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region
  • the antibody heavy chain constant region is conventional in the art, preferably a mouse-derived antibody heavy chain constant region or The human antibody heavy chain constant region, more preferably the human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a mouse-derived light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.
  • the heavy chain variable region represented by SEQ ID NO. 25, 33, 41, 49, 57, 65, 73, 81, 89 or 97 and the sequence are SEQ ID NO. 29, 37, 45, 53
  • the light chain variable region indicated by 61, 69, 77, 85, 93 or 101 can form a murine BlyS antibody and a human heavy chain constant region with a murine heavy chain constant region and a murine light chain constant region.
  • a human light chain constant region constitutes a BlyS chimeric antibody.
  • amino acid sequence of the above chimeric antibody is a sequence determined according to the Kabat definition in the heavy chain variable region represented by SEQ ID NO. 25, 33, 41, 49, 57, 65, 73, 81, 89 or 97.
  • Heavy chain CDRs as set forth in SEQ ID NO. 26-28, 34-36, 42-44, 50-52, 58-60, 66-68, 74-76, 82-84, 90-92 or 98-100
  • sequences according to the Kabat definition in the light chain variable region of the above sequence as SEQ ID NO. 29, 37, 45, 53, 61, 69, 77, 85, 93 or 101, such as SEQ ID NO.
  • Light chain CDRs shown at -40, 46-48, 54-56, 62-64, 70-72, 78-80, 86-88, 94-96 or 102-104 are each transplanted into a selected human germline template Substituting a CDR region of a human germline template, ie, a humanized antibody; the light chain framework region and the heavy chain framework region in the germline template are preferably selected from the human germline antibody heavy chain V, respectively, as described above H exon V H 1-18, outside the outer V H V H exon 3-7 and exon J H J H -6, and the human germline antibody light chain V K outer exon B3, J K outer exon V K and J K -4 exons A10.
  • a three-dimensional structure of the murine antibody based on the embedded residues, and CDR regions have direct interaction of residues, and has an important influence on the V H and V L, framework region conformation
  • the residue is subjected to a back mutation.
  • the amino acid sequence of the heavy chain variable region in which the CDR region is transplanted into the selected human germline template and the framework region residue is subjected to back mutation is preferably SEQ ID NO. 140, SEQ ID NO. ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146, SEQ ID NO. 149, SEQ ID NO. 150, SEQ ID NO. 151 or SEQ ID NO
  • the amino acid sequence of the light chain variable region is preferably SEQ ID No. 147, SEQ ID No. 148, SEQ ID No. 153, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156. Or as shown in SEQ ID No. 157.
  • the heavy chain variable region of SEQ ID NO. 1, 9 or 17 and the light chain variable region of SEQ ID NO. 5, 13 or 21 may be associated with a human antibody heavy chain constant region and human
  • the source antibody light chain constant region constitutes a fully human BlyS antibody.
  • the protein refers to an antibody protein, preferably, it is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multi-specific antibody, a single chain antibody fragment (scFv), a single One or more of a domain antibody (sdAb) and a single-region antibody (Signle-domain antibody), and a monoclonal antibody or a polyclonal antibody produced by the above antibody.
  • the monoclonal antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the full-length antibody protein is a conventional full-length antibody of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and the light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full human antibody full length protein.
  • the full length protein of the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, An Fd segment comprising a light chain variable region, a light chain constant region, and a heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab')2.
  • the single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.
  • the single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.
  • the preparation method of the protein is a conventional preparation method in the art.
  • the preparation method is preferably obtained by isolation from an expression transformant recombinantly expressing the protein or by artificially synthesizing a protein sequence.
  • the method for isolating the expression transformant which recombinantly expresses the protein is preferably obtained by cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain a recombinant expression.
  • the transformant can be isolated and purified by culturing the resulting recombinant expression transformant to obtain the protein.
  • the invention also provides a nucleic acid encoding the protein described above.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is SEQ ID No. 105, SEQ ID No. 107, SEQ ID No. 109, SEQ ID No. 111, SEQ ID No. 113, SEQ ID No. 115, SEQ ID No. 117, SEQ ID No. 119, SEQ ID No. 121, SEQ ID No. 123, SEQ ID No. 125, SEQ ID No. 127 or SEQ ID No. 129
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. 106, SEQ ID No. 108, SEQ ID No. 110, SEQ ID No. 112, SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 105 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the sequence listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 107, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in ID No. 106.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No.
  • nucleoside of the nucleic acid encoding the light chain variable region is represented by the sequence listing.
  • the acid sequence is shown in SEQ ID No. 110 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleic acid encoding the variable region of the light chain
  • the nucleotide sequence is shown in SEQ ID No. 112 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the Sequence Listing, and the light chain is variable.
  • nucleotide sequence of the nucleic acid of the region is shown in SEQ ID No. 114 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in the Sequence Listing SEQ ID.
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in No. 115, and the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in the sequence of SEQ ID No. 116.
  • SEQ ID No. 117 Listed as SEQ ID No. 117, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No.
  • nucleotide of the nucleic acid encoding the heavy chain variable region The sequence is shown in SEQ ID No. 119 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid of the chain variable region is shown in SEQ ID No. 121 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid of the heavy chain variable region is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 124 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 125 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 127, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the sequence listing.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 129 of the Sequence Listing, and the nucleoside encoding the nucleic acid of the light chain variable region The acid sequence is shown in SEQ ID No. 130 of the Sequence Listing.
  • the number in Table 2 is the sequence number in the sequence listing, such as the nucleotide sequence encoding the amino acid sequence of the heavy chain protein variable region of 2-1G11 is SEQ ID No. 105, and the light chain encoding 2-1G11 The nucleotide sequence of the amino acid sequence of the variable region of the protein is SEQ ID No. 106.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 2-1G11 is from positions 76 to 105 of SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 2-1G11 is in SEQ ID No. 105 of the Sequence Listing. 148th to 198th;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 2-1G11 is 295th to 342th in SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 2-1G11 is the 70th to 102nd positions in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence encoding CDR2 in the light chain protein variable region of 2-1G11 is 148th to 168th in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 2-1G11 is from position 265 to position 291 in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding L9G7 is from positions 76 to 105 of SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding L9G7 is 148th to 198th in SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding L9G7 is 295th to 342th in SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding L9G7 is from positions 70 to 102 of SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding L9G7 is 148th to 168th in SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding L9G7 is from position 265 to position 291 in SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding L1D12 is from positions 76 to 105 of SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding L1D12 is 148th to 195th in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding L1D12 is from position 292 to position 330 in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding L1D12 is from position 67 to position 99 in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding L1D12 is from positions 145 to 165 in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding L1D12 is 262th to 297th in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 35E6F7C3 is from positions 76 to 105 of SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 35E6F7C3 is from positions 148 to 198 of SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 35E6F7C3 is from positions 295 to 321 in SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 35E6F7C3 is from position 70 to position 99 of SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 35E6F7C3 is from positions 145 to 165 in SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 35E6F7C3 is 262th to 288th in SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 8E7D9C7F5 is from positions 76 to 105 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 8E7D9C7F5 is from positions 148 to 198 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 8E7D9C7F5 is from positions 295 to 342 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 8E7D9C7F5 is from positions 70 to 114 of SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 8E7D9C7F5 is from position 160 to position 180 in SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 8E7D9C7F5 is 277th to 303th in SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 20D1B6E9E5 is from positions 76 to 105 of SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 20D1B6E9E5 is 148th to 198th in SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 20D1B6E9E5 is SEQ ID No. of the Sequence Listing. 295th to 339th in 115;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 20D1B6E9E5 is from positions 70 to 117 of SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 20D1B6E9E5 is from position 163 to position 183 in SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 20D1B6E9E5 is from positions 280 to 306 in SEQ ID No. 116 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 78C11D2D12 is from positions 76 to 105 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 78C11D2D12 is from positions 148 to 198 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 78C11D2D12 is position 295 to 315 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 78C11D2D12 is from position 70 to position 99 of SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 78C11D2D12 is from positions 145 to 165 in SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 78C11D2D12 is 262th to 288th in SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 89A2G5E7 is from positions 76 to 105 of SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 89A2G5E7 is 148th to 195th in SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 89A2G5E7 is 292th to 327th in SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 89A2G5E7 is the 70th to 105th positions in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 89A2G5E7 is from positions 151 to 171 in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 89A2G5E7 is from positions 268 to 294 in SEQ ID No. 120 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 97E7B3F2 is from positions 76 to 105 of SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 97E7B3F2 is from positions 148 to 198 in SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 97E7B3F2 is from positions 295 to 321 of SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 97E7B3F2 is from positions 70 to 117 of SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 97E7B3F2 is from position 163 to position 183 in SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 97E7B3F2 is from positions 280 to 306 in SEQ ID No. 122 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 97A3C2H4 is from positions 76 to 105 of SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 97A3C2H4 is 148th to 198th in SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 97A3C2H4 is 295th to 327th in SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 97A3C2H4 is from positions 70 to 102 of SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 97A3C2H4 is 148th to 168th in SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 97A3C2H4 is from position 265 to position 291 in SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 67A2E1D10 is from positions 76 to 105 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 67A2E1D10 is from positions 148 to 198 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 67A2E1D10 is from positions 295 to 333 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 67A2E1D10 is SEQ ID No. 126 of the Sequence Listing. 70th to 102nd;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 67A2E1D10 is from positions 148 to 168 of SEQ ID No. 126 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 67A2E1D10 is position 265 to 291 of SEQ ID No. 126 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 111D10D6G3 is from positions 76 to 105 of SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 111D10D6G3 is 148th to 198th in SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 111D10D6G3 is from positions 295 to 309 of SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 111D10D6G3 is from positions 70 to 102 of SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 111D10D6G3 is 148th to 168th in SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 111D10D6G3 is from position 265 to position 291 in SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 93C6F10D3 is from positions 76 to 105 of SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 93C6F10D3 is 148th to 198th in SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 93C6F10D3 is from positions 295 to 336 of SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 93C6F10D3 is from positions 70 to 102 of SEQ ID No. 130 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 93C6F10D3 is from positions 148 to 168 of SEQ ID No. 130 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 93C6F10D3 is from position 265 to position 291 in SEQ ID No. 130 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the humanized antibody of the present invention engineered by the human framework region is SEQ ID NO. 158, SEQ ID NO .159, SEQ ID NO. 160, SEQ ID NO. 161, SEQ ID NO. 162, SEQ ID NO. 163, SEQ ID NO. 164, SEQ ID NO. 167, SEQ ID NO. 168, SEQ ID NO. 169 or SEQ ID NO.
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173, SEQ ID No. 174, or SEQ ID No. 175.
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial total sequence synthesis. .
  • the base sequence encoding the amino acid sequence of the above protein may be appropriately introduced with a substitution, deletion, alteration, insertion or addition to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more bases encoding a gene of the protein sequence within the range of activity of the antibody.
  • the invention also provides a recombinant expression vector comprising the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors.
  • the expression vector is a variety of vectors conventional in the art as long as it can accommodate the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell is E. coli TG1 or E. coli BL21 cells (expressing a single-chain antibody or Fab antibody), or HEK293 or CHO-K1 cells (expressing a full-length IgG antibody).
  • the recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.
  • the present invention also provides a method for producing a BLyS antibody, which comprises the steps of culturing the above recombinant expression transformant and obtaining a BLyS antibody from the culture.
  • the present invention also provides a method for detecting cells overexpressing BLyS protein, comprising the steps of: contacting the above-mentioned protein with a sample to be tested in vitro, and detecting the binding of the above protein to the sample to be tested.
  • overexpression refers to the overexpression of RNA or protein of the BLyS protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation), and due to protein Increased local overexpression and functional activity due to transport pattern changes (increased nuclear localization) (eg in substrates) The case of increased enzymatic hydrolysis).
  • the detection method of the above combination is a conventional detection method in the art, preferably a flow cytometry (FACS) test.
  • FACS flow cytometry
  • the present invention provides a composition for detecting a cell overexpressing a BLyS protein, which comprises the above protein as an active ingredient.
  • a composition for detecting a cell overexpressing a BLyS protein which comprises the above protein as an active ingredient.
  • it further comprises a compound consisting of functional fragments of the above proteins as an active ingredient.
  • the present invention provides the use of the above proteins for the preparation of a medicament.
  • the medicament is a medicament for preventing or treating a disease associated with abnormal expression or dysfunction of BLyS.
  • the disease associated with abnormal expression or function of BLyS is a disease conventionally associated with abnormal expression or function of BLyS in the art.
  • it is an autoimmune disease, an inflammatory disease, an infectious disease or a proliferative disease.
  • the autoimmune disease is a conventional autoimmune disease in the art, preferably lupus erythematosus (SLE), rheumatoid arthritis (RA), dry syndrome (SS), multiple sclerosis (MS). , myasthenia gravis (MG), chronic thyroiditis or immunodeficiency syndrome.
  • the inflammatory disease is a inflammatory disease conventional in the art, preferably asthma or an allergic disease.
  • the infectious disease is a conventional infectious disease in the art.
  • it is acquired immunodeficiency syndrome (AIDS).
  • the proliferative disease is a conventional proliferative disease in the art, preferably leukemia, tumor or lymphoma.
  • the present invention also provides a pharmaceutical composition comprising the above-mentioned protein as an active ingredient.
  • the pharmaceutical composition is a pharmaceutical composition for preventing or treating a disease associated with abnormal expression or dysfunction of BLyS.
  • the administration route of the pharmaceutical composition of the present invention is preferably administered by injection or orally.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is in various forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule. , injection or infusion, etc.
  • the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutically acceptable carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above protein and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being a percentage by mass of the pharmaceutical composition.
  • the pharmaceutical composition is administered in an amount effective to reduce or delay the progression of a disease, degenerative or damaging condition.
  • the effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the results sought.
  • One skilled in the art can determine the effective amount by using the above factors such as the individual basis and using no more than conventional experiments.
  • the present invention provides the use of the above protein for the preparation of a medicament for preventing or treating a disease associated with abnormal expression or dysfunction of BLyS.
  • the disease associated with abnormal expression or function of BLyS is an autoimmune disease, an inflammatory disease, an infectious disease or a proliferative disease.
  • the present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for preventing or treating a disease associated with abnormal expression or dysfunction of BLyS.
  • the disease associated with abnormal expression or function of BLyS is an autoimmune disease, an inflammatory disease, an infectious disease or a proliferative disease.
  • the reagents and starting materials used in the present invention are commercially available.
  • a positive progressive effect of the present invention is that the protein of the present invention is a humanized or fully human BLyS antibody which has a high affinity with the BLyS protein (affinity KD ⁇ 5*10 -9 M) and is capable of protein level And the cell level effectively blocks the BLyS protein and prevents the binding of the BLyS protein to the receptor.
  • the BLyS antibody lacks cross-reactivity with a similar protein antigen such as human APRIL.
  • the B cell proliferation assay demonstrated that the BLyS antibody has good biological activity and can inhibit the proliferation of mouse B cells induced by human BLyS.
  • the BLyS antibodies alone or in combination with other drugs can be used to detect, diagnose, treat or screen for diseases associated with abnormal expression or dysfunction of BLyS, such as autoimmune diseases, inflammatory diseases, infectious diseases or proliferative diseases.
  • Figure 1 is a graph showing the results of biological activity assay of purified hBLyS-ECD. Where RLU represents the relative light unit.
  • 2A and 2B are graphs showing the results of detecting the expression level of the CHO-K1 recombinant cell line BLyS protein by flow cytometry.
  • 2C4 represents the clone number
  • 4C4 represents the clone number
  • the antibody refers to the BLyS antibody (purchased from eBioscience)
  • the negative control refers to the isotype antibody control.
  • Figure 3 is a graph showing the results of serum antibody titers of BALB/c and SJL mice after ELISA for immunogen immunization.
  • Figure 4 is a graph showing the results of enzyme-linked immunosorbent assay for detecting the activity of biotinylated hBLyS-ECD.
  • 5A and 5B are graphs showing the results of the reactivity of BLyS purified antibodies with biotinylated hBLyS-ECD in an enzyme-linked immunosorbent assay.
  • 6A and 6B are diagrams showing the results of an enzyme-linked immunosorbent assay for detecting the reaction of a BLyS purified antibody with a BLyS homologous APRIL protein.
  • Figure 7 shows the results of flow cytometry analysis of the reactivity of BLyS purified antibody with human BLyS recombinant cells.
  • Figure 8 is a graph showing the results of a flow cytometric assay for detecting the reactivity of BLyS purified antibody with monkey BLyS recombinant cells.
  • Figure 9A and Figure 9B are graphs showing the results of binding activity of BLyS purified antibody blocking BLyS receptor BAFF R to biotinylated hBLyS-ECD.
  • Figure 10 is a graph showing the results of inhibition of proliferation of mouse B cells stimulated by hBLyS-ECD by BLyS purified antibodies.
  • Figure 11 is a graph showing the results of inhibition of proliferation of mouse B cells stimulated by murine BLyS by BLyS purified antibodies.
  • Figure 12 is a graph showing the results of the reactivity of the BLyS chimeric antibody with biotinylated hBLyS-ECD in an enzyme-linked immunosorbent assay.
  • Figure 13 is a graph showing the results of blocking the binding activity of the BLyS receptor BAFF R to biotinylated hBLyS-ECD by the BLyS chimeric antibody.
  • Figure 14 is a graph showing the results of inhibition of proliferation of mouse B cells stimulated by hBLyS-ECD by BLyS chimeric antibodies.
  • 15A to 15C are graphs showing the effect of the BLyS chimeric antibody and the fully human antibody on the proportion of mouse B cells stimulated by hBLyS-ECD in splenocytes.
  • Figure 16 is a graph showing the results of the reactivity of BLyS humanized antibody with biotinylated hBLyS-ECD in an enzyme-linked immunosorbent assay.
  • Figure 17 is a graph showing the results of binding activity of BLyS humanized antibody blocking BLyS receptor BAFF R to biotinylated hBLyS-ECD.
  • Figure 18 is a graph showing the results of inhibition of proliferation of mouse B cells stimulated by hBLyS-ECD by BLyS humanized antibodies.
  • 19A to 19B are graphs showing the effects of BLyS humanized antibodies on the proportion of mouse B cells stimulated by hBLyS-ECD in splenocytes and the level of IgA in serum.
  • room temperature as used in the examples means the temperature between the operations to be tested, and is generally 15 to 30 °C.
  • HEK293 cells purchased from Invitrogen for transient transfection (PEI, Polysciences), using FreeStyle TM 293 (commercially available from Invitrogen) were expanded at 37 °C. After 7 days, the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing the extracellular region of the human BLyS protein. The culture supernatant was applied to a Ni affinity chromatography column (purchased from GE Healthcare) while monitoring the change in ultraviolet absorption value (A 280 nm ) with an ultraviolet (UV) detector.
  • a 280 nm ultraviolet absorption value
  • the Ni affinity chromatography column was washed with phosphate buffer (pH 7.4) containing 5% (v/v) sucrose and 0.01% (v/v) Tween-80 until the UV absorption value returned to the baseline, then Gradient elution with 0-500 mM imidazole.
  • the His-tagged human BLyS protein extracellular region eluted from the Ni affinity column was collected using a phosphate buffer containing 5% (v/v) sucrose and 0.01% (v/v) Tween-80. (pH 7.4) Dialysis and protein concentration at 4 ° C in an ultrafiltration tube.
  • the dialyzed protein was sterile-filtered at 0.22 ⁇ m and stored at -80 ° C to obtain a purified human extracellular region of BLyS protein as an immunogen (ie, hBLyS-ECD).
  • the immunogen is subjected to a series of quality control tests before use, such as detecting protein concentration, purity, molecular weight, biological activity, and the like.
  • the biological activity of the immunogen is detected by a B cell proliferation assay (see Example 5 for the method).
  • the immunogen can stimulate the proliferation of mouse B cells.
  • the 132th position of the amino acid sequence of human or monkey BLyS (ie hBLyS or cyno BLyS) (gene accession number in NCBI is Q9Y275, EHH58704.1, respectively) was mutated from arginine to histidine to stabilize the construction.
  • the cell line expresses intact human or monkey BLyS without cleavage.
  • the nucleotide sequence of the BLyS full-length amino acid sequence of the mutated human or monkey (as shown in SEQ ID No. 132 and SEQ ID No. 133 of the sequence listing) was cloned into the pLVX vector (purchased from Clontech) by Shanghai Ji Ma Pharmaceutical Technology Co., Ltd.
  • the amplified clones were further screened by flow cytometry using a known BLyS antibody (purchased from eBioscience).
  • the cell line with good growth and high fluorescence intensity is selected, and the monoclonal cell line continues to expand and cryopreservation, and a stable cell line expressing human or monkey BLyS is obtained.
  • the specific selection results are shown in Table 4 and Figures 2A to 2B.
  • the positive cells (%) in Table 4 refer to the percentage of positive cells in the total number of cells. Table 4 illustrates that a series of BLYS positively expressed CHO-K1 cell lines have been made.
  • the immunogen obtained in the step (1) i.e., hBLyS-ECD
  • the immunogen obtained in the step (1) was emulsified with Freund's complete adjuvant and intraperitoneally injected with 0.2 mL, i.e., 100 ⁇ g of immunogen was injected per mouse.
  • the immunogen was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.2 mL, that is, 50 ⁇ g of immunogen was injected per mouse.
  • mice B. 6-8 weeks old female BALB/c and SJL mice (purchased from Shanghai Slack Laboratory Animals Co., Ltd.) were used, and the mice were raised under SPF conditions.
  • the mice were injected with 25 ⁇ L of the ankle joint after mixing the immunogen (hBLyS-ECD), oligonucleotide (CpG) and GERBU adjuvant, that is, each mouse was injected with 10 ⁇ g of immunogen.
  • the immunogen hBLyS-ECD
  • CpG oligonucleotide
  • GERBU adjuvant that is, each mouse was injected with 10 ⁇ g of immunogen.
  • 25 ⁇ L of the ankle joint was injected after the immunogen and GERBU adjuvant were mixed, that is, 5 ⁇ g of the immunogen was injected per mouse.
  • ⁇ g or 5 ⁇ g of immunogen was injected into the abdominal cavity or ankle joint of each selected and last immunized mouse, and the mice were sacrificed 5 days later, and spleen cells were collected.
  • NH 4 OH was added to a final concentration of 1% (w/w), and the erythrocytes mixed in the spleen cells were lysed to obtain a spleen cell suspension.
  • the cells were washed three times with DMEM basal medium (purchased from invitrogen) at 1000 rpm, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 5:1 viable cells, using high-efficiency electrofusion method.
  • the fused cells were diluted into DMEM medium containing 20% (w/w) fetal bovine serum and 1 ⁇ HAT. Then, 1 ⁇ 10 5 / 200 ⁇ L per well was added to a 96-well cell culture plate, and placed in a 5% (v/v) CO 2 , 37 ° C incubator. After 14 days, the cell fusion plate was screened by indirect ELISA, and the positive clone with OD 450nm > 1.0 in the ELISA was amplified into 24-well plates in DMEM containing 10% (w/w) HT fetal bovine serum at 37 ° C, 5%.
  • hybridoma cells with OD 450nm >1.0 in the ELISA assay and hybridoma cell culture supernatant in the ligand receptor binding assay with a blocking inhibition rate of 60% of the BLyS receptor were selected as positive clones.
  • the selected hybridoma cells were subcloned in a 96-well plate by limiting dilution in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) Culture under CO 2 conditions.
  • a preliminary screening was performed by ELISA 10 days after subcloning, and a single positive monoclonal was selected and expanded into a 24-well plate to continue the culture.
  • Bioactivity was assessed 3 days later using receptor ligand binding assays.
  • the evaluation standard was OD 450nm >1.0 in the ELISA experiment, and the blocking inhibition rate of the BLyS receptor in the hybridoma cell culture supernatant in the ligand receptor binding experiment reached 60%.
  • the best clones were selected and cultured in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) CO 2 conditions.
  • the optimal clone is expanded and cultured, and the liquid nitrogen is frozen to obtain the optimal hybridoma cells, and can be used for subsequent antibody production and purification.
  • the concentration of the antibody produced by the hybridoma cells is low, only about 1 to 10 ⁇ g/mL and the concentration of the obtained antibody varies greatly; in addition, the various proteins produced by the cell culture in the medium and the fetal bovine serum component of the medium are Many biological activity assays have varying degrees of interference, so small-scale (1 to 5 mg) antibody production purification is required.
  • the hybridoma cells obtained in the step (III) were inoculated into a T-75 cell culture flask and domesticated for 3 passages using a production medium (Hybridoma serum free medium, available from Invitrogen). After the growth state is good, inoculate the cell culture spinner. 500 mL of production medium was added to each 2 liter culture spinner flask, and the inoculated cell density was 1.0 ⁇ 10 5 /mL. The cap was capped and the roller was placed in a bottle shaker in a 37 ° C incubator at 3 rpm. After continuous rotation culture for 14 days, the cell culture medium was collected, and the cells were removed by filtration, and filtered through a 0.45 ⁇ m filter to clarify the culture supernatant. The clarified culture supernatant can be purified immediately or frozen at -30 °C.
  • a production medium Hybridoma serum free medium, available from Invitrogen
  • the monoclonal antibody in the clarified culture supernatant of 300 mL of hybridoma cells was purified using a 2 mL Protein G column (purchased from GE Healthcare).
  • the Protein G column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2) and the clarified culture supernatant was then loaded onto a Protein G column with a flow rate of 3 mL/min. After loading, the protein G column was washed with equilibration buffer, and the volume of the equilibration buffer was 4 times the volume of the column of the protein G column.
  • equilibration buffer PBS phosphate buffer, pH 7.2
  • the monoclonal antibody bound to the Protein G column was eluted with an eluent (0.1 M glycine hydrochloride buffer, pH 2.5), and the elution condition (A280 ultraviolet absorption peak) was monitored by a UV detector.
  • the eluted monoclonal antibody was collected and neutralized by adding 10% (v/v) 1.0 M Tris-HCl buffer. Immediately thereafter, it was dialyzed against PBS phosphate buffer overnight, and the next day, the solution was changed once and dialysis was continued for 3 hours.
  • the dialyzed monoclonal antibody was collected, sterile-filtered with a 0.22 ⁇ m filter, and stored aseptically to obtain a purified BLyS antibody as a lead antibody.
  • the purified BLyS antibody was subjected to detection analysis such as protein concentration (A280/1.4), purity, and endotoxin (Lonza kit), and the results are shown in Table 6. As a result, it was found that the endotoxin concentration of the lead antibody was within 1.0 EU/mg.
  • Biotin-XX-NHS purchased from Sigma Aldrich
  • hBLyS-ECD the immunogen prepared in Example 1
  • hBLyS-ECD the immunogen prepared in Example 1
  • the free biotin was then removed by dialysis against PBS phosphate buffer overnight to obtain a biotinylated immunogen (i.e., biotinylated hBLyS-ECD).
  • concentration of biotinylated hBLyS-ECD was determined using a BCA protein concentration assay kit (purchased from Pierce).
  • biotinylated hBLyS-ECD The activity of biotinylated hBLyS-ECD was determined by ELISA.
  • BLyS antibody purchased from GSK
  • ELISA microplate 50 ⁇ L/well
  • ELISA microplate 50 ⁇ L/well
  • ELISA microplate 50 ⁇ L/well
  • ELISA microplate 50 ⁇ L/well
  • ELISA microplate 50 ⁇ L/well
  • ELISA containing 1% (w/v) BSA and 0.05% (v /v) Tween-20 in PBS phosphate buffer, pH 7.4
  • Streptavidin-labeled horseradish peroxidase (purchased from Sigma, product number S5512) was added and incubated for 30 minutes at room temperature. After adding 100 ⁇ L/well of TMB color developing solution and incubating for 15 minutes at room temperature, the color reaction was terminated by adding 5 ⁇ L of 1N hydrochloric acid, and the OD 450 nm reading was read by an ELISA plate reader. The results are shown in Figure 4 and Table 7. The results indicate that biotinylated hBLyS-ECD can bind to BLyS antibodies.
  • the leader antibody was screened using a native human single-chain antibody (ScFv) phage display library (constructed by Shanghai Ruizhi Chemical Research Co., Ltd.). Antibodies that bind to BLyS were obtained by four rounds of biopanning. The specific process is as follows:
  • Streptavidin was diluted to 12.5 ⁇ g/mL with PBS, added to an immunotube at 1 mL per tube, and incubated overnight at 4 °C. After the immunotubes were washed 3 times with PBS, 50 ⁇ g of the biotinylated hBLyS-ECD prepared in the step (1) was added to each of the half of the immunotubes, and shaken at room temperature for 1 hour. After washing 3 times with PBS buffer, the immunotubes were blocked with 10 mL of 2% (w/v) blocking solution [PBS buffer containing 2% (w/v) skim milk powder] for 2 hours at room temperature.
  • 2YT medium was prepared by adding 10 g of yeast extract, 16 g of tryptone and 5 g of NaCl to 1 L of water, and then adjusting the pH to 7.0 with NaOH, autoclaving). Collect and inoculate into fresh medium and incubate at 37 ° C until log phase.
  • the helper phage M13KO7 (purchased from NEB, item number N0315S) was added, mixed, and allowed to stand at 37 ° C for 30 minutes.
  • the cells were cultured by shaking at 37 ° C for 30 minutes, centrifuged at 4000 rpm for 10 minutes, and the cells were collected, and fresh medium was added thereto, and cultured at 30 ° C for 4 hours with shaking. After centrifugation at 4000 rpm for 30 minutes, the supernatant was collected, and a volume of 1/4 volume of a 2.5 M NaCl solution containing 5 x PEG was added to the supernatant volume, and placed on ice overnight. After centrifugation at 4000 rpm for 30 minutes at 4 ° C, the phage pellet was collected and dissolved in PBS buffer. The residual cell debris was removed by centrifugation at 10,000 rpm for 10 minutes, and the supernatant was collected for the next round of biopanning.
  • the scFv antibody obtained by the screening was confirmed to have binding activity to the BLyS protein by an ELISA method. Clones with OD 450 nm > 1.0 were picked for sequencing to obtain clones with different HCDR3 sequences. Then, by FACS and ligand receptor binding experiments, clones with MFI values >30 in FACS experiments and 60% inhibition of BLyS receptors in cell-cleaved supernatants in ligand receptor binding assays were selected as eligible. Positive clone.
  • primers were designed (specific primer sequences are shown in Table 8), and the light chain and heavy chain variable regions were separately amplified by a PCR method.
  • a 50 ⁇ L reaction system was configured, including 0.5 ⁇ L of the plasmid containing the transfected positive clone Escherichia coli TG1, 10 pmol of each primer, 0.5 ⁇ L of DNA polymerase, and a matching buffer system.
  • the PCR program was set up, pre-denatured at 95 ° C for 2 minutes, denatured at 95 ° C for 15 seconds, annealed at 55 ° C for 30 seconds, extended at 68 ° C for 45 seconds, and further extended at 68 ° C for 10 minutes after 30 cycles to obtain a PCR product.
  • the DNA polymerase used for PCR was purchased from Invitrogen, Cat. No. 12344; the buffer system was a buffer system purchased for the DNA polymerase. 5 ⁇ L of the PCR product was detected by agarose gel electrophoresis, and the positive sample was purified using a column recovery kit, wherein the recovery kit was Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609.
  • the ligation reaction was carried out: inserting 3 ⁇ L of the fragment, 0.5 ⁇ L of the digested expression vector, 0.5 ⁇ L of the recombinant enzyme Exnase, 2 ⁇ L of the buffer solution, and 10 ⁇ L of the reaction system, and reacting at 37 ° C for half an hour to obtain a ligation product, that is, a constructed recombinant vector.
  • the recombinant enzyme was purchased from Vazyme, Cat. No. C112-01/02; the buffer was the recombinant enzyme purchased for the recombinant enzyme; the heavy chain variable region was directionally cloned into the constant region of the IgG1 containing the signal peptide and the human antibody heavy chain.
  • Expression vector (wherein the expression vector was purchased from Invitrogen, the recombination step was completed by Shanghai Ruizhi Chemical Research Co., Ltd.), and the light chain variable region was directionally cloned into the light chain kappa or lambda containing the signal peptide and human antibody (only for the following L1D12 antibody)
  • the light chain) constant region expression vector (where the expression vector was purchased from Invitrogen and the recombination step was performed by Shanghai Ruizhi Chemical Research Co., Ltd.). 10 ⁇ L of the ligation product was added to 50 ⁇ L of competent cells (Ecos101competent cells, available from Yeastern, Cat. No. FYE607), and ice-bathed for 30 minutes.
  • the colony PCR system was: 1 ⁇ L of each primer, 10 ⁇ L of PCR master mix (purchased from Novoprotein), and made up to 20 ⁇ L with water. The pipettes were pipetted into the PCR reaction system, and 0.5 ⁇ L of the colony was aspirated to another LB solid culture dish containing 100 ⁇ g/mL ampicillin to preserve the strain. After the end of the PCR reaction, 5 ⁇ L was taken for agarose gel electrophoresis detection, and the positive samples were sequenced and analyzed [see Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)].
  • the recombinant antibody light and heavy chains were transiently transfected into FreeStyle TM 293-F cells (purchased from Invitrogen) for production of the antibody.
  • the density of 293-F cells should be 1-1.5 ⁇ 10 6 /mL, and 100 ⁇ g of the above-prepared vector DNA is required for 100 mL of cells (wherein the mass ratio of recombinant light chain vector and heavy chain vector) 3:2) and 200 ⁇ g of transfection reagent polyethyleneimine (PEI).
  • the vector DNA and PEI were separately added to the medium, allowed to stand at room temperature for 5 minutes, and filtered through a 0.22 ⁇ m filter, and the mixture was mixed with PEI and allowed to stand at room temperature for 15 minutes. The mixture was then slowly added to the cells and incubated at 120 ° C in an 8% (v/v) CO 2 incubator at 120 rpm. On the second day after transfection, 0.5% (v/v) peptone was added to the cell culture medium. After 6-7 days, 3500 g of the culture medium of the cells was centrifuged for 30 minutes, and the supernatant was collected and filtered through a 0.22 ⁇ m filter.
  • Monoclonal antibodies in 200 mL of clarified supernatant were purified using a 1 mL Protein A column (available from GE Healthcare).
  • the Protein A column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2) and the supernatant was loaded onto a Protein A column with a flow rate of 3 mL/min. After loading, the protein A column was washed with equilibration buffer, and the volume of the equilibration buffer was 5 times the volume of the column of the protein A column.
  • equilibration buffer PBS phosphate buffer, pH 7.2
  • the BLyS antibody bound to the Protein A column was eluted with an eluent (0.1 M glycine hydrochloride buffer, pH 3.0), and the elution condition (A280 ultraviolet absorption peak) was monitored with a UV detector.
  • the eluted antibody was collected, neutralized by adding 10% (v/v) 1.0 M Tris-HCl buffer, and immediately dialyzed overnight with PBS phosphate buffer, and the solution was changed once a day for another 3 hours.
  • the dialysis BLyS antibody was collected, sterile-filtered with a 0.22 ⁇ m filter, and stored as a sterile antibody to obtain a purified BLyS antibody as a lead antibody.
  • the lead antibody was assayed for protein concentration (A280/1.4), purity, and endotoxin (Lonza kit). The results are shown in Table 9, and the results showed that the lead antibody endotoxin concentration was within 1.0 EU/mg.
  • ELISA enzyme-linked immunosorbent assay
  • the leader antibodies obtained in Examples 1 and 2 were subjected to cross-reactivity with human APRIL proteins of the family of human BLyS protein and BLyS protein, respectively.
  • Streptavidin was first diluted to a final concentration of 1.0 ⁇ g/mL with PBS, and then added to a 96-well ELISA plate at 50 ⁇ L per well, and incubated at 4 ° C overnight with a plastic membrane. The next day, the plate was washed 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20], and a blocking solution [containing 0.05% (v/v) Tween 20 and 1% (w/w) BSA was added. PBS buffer], blocked at 37 ° C for 2 hours.
  • the blocking solution was discarded, and the biotinylated hBLyS-ECD obtained in Example 2 was diluted with PBS to a final concentration of 50 ng/mL, and then added to a 96-well ELISA plate at 50 ⁇ L per well, and incubated at 37 ° C for 1 hour. After washing the plate 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20], 100 ⁇ L of the purified leader antibody obtained in Examples 1 and 2 was added to each well. After incubating for 1 hour at 37 ° C, the plate was washed 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20].
  • the APRIL protein purchased from R&D Systems
  • PBS buffer containing 0.05% (v/v) Tween 20
  • a blocking solution [containing 0.05% (v/v) Tween 20 and 1% (w/w) BSA was added.
  • PBS buffer blocked at 37 ° C for 2 hours.
  • the blocking solution was discarded, and 100 ⁇ L of the lead antibody obtained in Examples 1 and 2 was added per well. After incubating for 1 hour at 37 ° C, the plate was washed 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20]. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma) was added, and after incubation at 37 ° C for 1 hour, the plate was washed 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20]. .
  • the A 450 nm value was read by an ELISA plate reader (SpectraMax M5e, purchased from Molecular Device), and the results are shown in Figures 6A to 6B and Table 11, and Table 11 shows that the APRIL recombinant protein of the same family of the lead antibody and BLyS protein was in ELISA. Levels are not combined.
  • the IgG control was human IgG and the data in the table is the OD 450 nm value.
  • the CHO-K1hBLyS stable cell strain obtained in the step (2) of Example 1 and the CHO-K1cynoBLyS stable cell strain were expanded to a 90% confluence in a T-175 cell culture flask. Exhaustion medium, with PBS buffer (commercially available from Invitrogen) were washed once and then dissociated with a cell solution (TrypLE TM Express Enzyme, available from Life technology company) and treated cells were collected. The specific steps are: washing the cells once with PBS buffer, performing cell counting, diluting the cells with PBS buffer to 2 ⁇ 10 6 cells per ml, adding 2% (w/w) fetal bovine serum blocking solution, on ice. Incubate for 30 minutes.
  • Tables 12-13 show that the leader antibody binds to the human BLyS protein on the cell surface, and the lead antibody and the monkey BLyS cross-react.
  • the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • the affinity constant was determined using a Biacore X100 instrument (available from GE Healthcare). The specific operations and methods are based on the instrument manual and the detailed methods provided by the manufacturer. Specifically, the affinity measurement was performed using a CM5 chip (Sensor Chip CM5, available from GE Healthcare). The CM5 chip was first activated with 50 mM NaOH and 50 mM NHS and 200 mM EDC mixed in a volume ratio of 1:1, and then the anti-human Fc fragment antibody (purchased from Genway) was diluted with 10 mM sodium acetate buffer (pH 5.0). The coupling reaction with the CM5 chip was carried out to 16.4 ⁇ g/ml. The remaining activation sites were then blocked by the injection of 1 M ethanolamine.
  • Example 2 After the antibody to be tested (that is, the fully human BLyS antibody prepared in Example 2) was captured by the chip, 7 different concentrations of the immunogen prepared in Example 1 (ie, hBLyS-ECD) were injected, and the antibody was detected by a Biacore instrument. Antigen binding and dissociation. The dissociation constant and binding constant were then fitted using Biacore X100 Evaluation Software 2.0 software. The affinity constant is the ratio of the dissociation constant to the binding constant. The results are shown in Table 14, indicating that the BLyS antibody has high affinity for hBLyS-ECD.
  • BLyS antibodies blocked the binding of BLyS protein to its receptor BAFF R by receptor ligand binding assay of BLyS protein.
  • BAFF R (purchased from R&D Systems) was added to a 96-well ELISA plate at 50 ng per well, and the plastic film was incubated overnight at 4 °C. The next day, the plate was washed 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20], and a blocking solution [containing 0.05% (v/v) Tween 20 and 2% (w/w) BSA was added. PBS buffer] was blocked at room temperature for 1 hour.
  • Example 5 Mouse B cell proliferation assay to detect the effect of BLyS purified antibody on the proliferation of human or murine BLyS protein in B cells
  • Freshly obtained mouse spleens were ground, resuspended in RPMI1640 medium (purchased from Invitrogen, Cat. No. A10491) containing 10% (w/w) fetal bovine serum, and filtered through a 70 ⁇ m cell strainer (purchased from BD). Centrifuge at 1,500 rpm for 4 minutes at 4 °C. The supernatant was discarded, and the cell pellet was vortexed for 15 seconds, and red blood cell lysis buffer (purchased from Sigma) was added, and allowed to stand at room temperature for 5 minutes.
  • the supernatant was supplemented by RPMI1640 medium containing 10% (w/w) fetal bovine serum to 15 mL, and centrifuged at 1500 rpm for 4 minutes at 4 °C.
  • the supernatant was filtered with a 40 ⁇ m cell strainer (purchased from BD) to obtain a filtered supernatant, followed by cell counting.
  • the mouse B cells were isolated using a kit (purchased from Miltenyi Biotec), and the experimental procedures were strictly in accordance with the kit instructions. The specific experiment is briefly described as follows: The filtered supernatant was centrifuged at 300 g for 4 minutes at 4 ° C, and the supernatant was discarded.
  • the LS separation column (purchased from Miltenyi Biotec) was placed on a magnetic stand, first added with 3 mL of buffer rinse, and then the cell suspension was added to collect the flow through, ie, the unlabeled enriched B cell suspension. Further, 3 mL of the buffer solution was added, and the flow-through solution was collected and combined with the flow-through solution of the previous step to obtain B cells isolated from the mouse spleen.
  • the B cells obtained in the step (1) of Example 5 were plated to a 96-well cell culture plate at 50 ⁇ L per well of 5 ⁇ 10 4 cells, and 2 ⁇ g/ml of F(ab') 2 fragmented goat anti-mouse IgM secondary antibody was added. (purchased from Jackson ImmunoResearch), 10 ng/ml of the immunogen (hBLyS-ECD) prepared in Example 1 or the murine BLyS protein (purchased from R&D Systems) and different concentrations of the lead antibody prepared in Example 1 or 2 were ensured. Each reaction well has a volume of 100 ⁇ L. The number of viable cells was measured after incubating the reaction plate at 37 ° C for 72 hours in a 5% (v/v) CO 2 incubator.
  • Luminescent Cell Viability Assay kit purchased from Promega
  • the specific experiment is briefly described as follows: 96-well plates were equilibrated at room temperature for 30 minutes before the assay, and the volume was equal to the cell culture medium. The reagent was placed on a shaker for 2 minutes to induce cell lysis, and the well plate was placed at room temperature for 10 minutes to stabilize the luminescence signal, and finally the value was read with a plate reader (SpectraMax 384plus, Molecular Device).
  • the purified whole human BLyS antibody and hybridoma antibody were subjected to competitive enzyme-linked immunosorbent assay to analyze the epitopes bound by different antibodies.
  • the purified BLyS antibody was first diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well ELISA plate at 50 ⁇ L per well, and incubated at 4 ° C overnight with a plastic membrane. The next day, the plate was washed 4 times with a washing solution [PBS buffer containing 0.05% (v/v) Tween 20], and a blocking solution [containing 0.05% (v/v) Tween 20 and 1% (w/w) BSA was added. PBS buffer], blocked at 37 ° C for 1 hour.
  • the blocking solution was discarded, and the competitive antibody and IgG control were diluted with PBS to a final concentration of 40 ⁇ g/mL, and then added to a 96-well ELISA plate at 50 ⁇ L per well.
  • the biotinylated hBLyS-ECD was then diluted with PBS to a final concentration of 2 ng/mL and then added to a 96-well ELISA plate at 50 [mu]L per well.
  • the final concentrations of competitive antibodies and hBLyS-ECD were guaranteed to be 20 ⁇ g/mL, 1 ng/mL, respectively.
  • A450 nm values were read using an ELISA plate reader (SpectraMax M5e, available from Molecular Device).
  • the results are shown in Tables 18A to 18B, indicating that there are different degrees of competition between BLyS antibodies, that is, binding to different antigenic epitopes.
  • the data in the table is the inhibition rate (%) of the binding level of the original antibody to hBLyS-ECD after the addition of the competitive antibody.
  • Total RNA isolation The supernatant obtained by subcloning culture corresponding to the lead antibody selected in Example 1 was tested for antigen binding (i.e., after the assays and activity assays of Examples 3 to 5), and 5 ⁇ was collected by centrifugation.
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ l of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated at 85 ° C for 10 minutes.
  • a 50 ⁇ L PCR system was configured, including 1 ⁇ L of cDNA, 25 pmol of each primer, 1 ⁇ L of DNA polymerase, and a matching buffer system, 250 ⁇ mol dNTPs; a PCR program was set, predenatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, and annealed at 55 ° C for 30 seconds.
  • the extension was carried out at 72 ° C for 35 seconds, and after 35 cycles, an additional 5 minutes at 72 ° C to obtain a PCR product.
  • the kit for reverse transcription was PrimeScript RT Master Mix, purchased from Takara, Catalog No. RR036; the kit used for PCR included Q5 super-fidelity enzyme, available from NEB, Cat. No. M0492.
  • PCR product 5 ⁇ L was detected by agarose gel electrophoresis, and the positive samples were purified using a column recovery kit, wherein the recovery kit was Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609.
  • the ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 ⁇ L of ligase, 1 ⁇ L of buffer, 10 ⁇ L of reaction system, and the reaction product was ligated at 16 ° C for half an hour to obtain a ligation product, wherein the ligated kit was T4 DNA ligase, purchased from NEB, and the product number was M0402.
  • 5 ⁇ L of the ligation product was added to 100 ⁇ L of competent cells (Ecos 101competent cells, purchased from Yeastern, item number FYE607), ice bathed for 5 minutes, then heat-shocked in a 42 ° C water bath for 1 minute, put back on ice for 1 minute, and then added 650 ⁇ L.
  • the antibiotic SOC medium was incubated at 37 RPM for 30 minutes on a 37 ° C shaker, and 200 ⁇ L of the solution was applied to an antibiotic-containing LB solid medium and incubated at 37 ° C overnight; the next day, the T-carrier primers M13F and M13R were used.
  • the 30 ⁇ L PCR system was configured, colony PCR was performed, and the colony was pipetted in a PCR reaction system with a pipette tip, and 0.5 ⁇ L of the spot was sucked onto another LB solid culture dish containing 100 ⁇ g/mL ampicillin to preserve the strain; After the end of the PCR reaction, 5 ⁇ L was taken for agarose gel electrophoresis, and the positive samples were sequenced and analyzed [see Kabat, "Sequences of Proteins of Immunological Interest," National Ins Titutes of Health, Bethesda, Md. (1991)]. The sequencing results are shown in Tables 19-20.
  • the number in Table 19 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of 2-1G11 is SEQ ID No. 1, and the CDR1 of the heavy chain protein variable region of 2-1G11 The amino acid sequence is SEQ ID No. 2.
  • the number in Table 20 is the sequence number in the sequence listing, such as the nucleotide sequence of the amino acid sequence encoding the heavy chain protein variable region of 2-1G11 is SEQ ID No. 105, and the light chain encoding 2-1G11 The nucleotide sequence of the amino acid sequence of the variable region of the protein is SEQ ID No. 106.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 2-1G11 is from positions 76 to 105 of SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 2-1G11 is from positions 148 to 198 of SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 2-1G11 is 295th to 342th in SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 2-1G11 is the 70th to 102nd positions in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence encoding CDR2 in the light chain protein variable region of 2-1G11 is 148th to 168th in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 2-1G11 is from position 265 to position 291 in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding L9G7 is from positions 76 to 105 of SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding L9G7 is 148th to 198th in SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding L9G7 is 295th to 342th in SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding L9G7 is from positions 70 to 102 of SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding L9G7 is in SEQ ID No. 108 of the Sequence Listing 148th to 168th;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding L9G7 is from position 265 to position 291 in SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding L1D12 is from positions 76 to 105 of SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding L1D12 is 148th to 195th in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding L1D12 is from position 292 to position 330 in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding L1D12 is from position 67 to position 99 in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding L1D12 is from positions 145 to 165 in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding L1D12 is position 262 to 297 in SEQ ID No. 110 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 35E6F7C3 is from positions 76 to 105 of SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 35E6F7C3 is from positions 148 to 198 of SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 35E6F7C3 is from positions 295 to 321 in SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 35E6F7C3 is from position 70 to position 99 of SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 35E6F7C3 is from positions 145 to 165 in SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 35E6F7C3 is position 262 to 288 in SEQ ID No. 112 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 8E7D9C7F5 is from positions 76 to 105 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 8E7D9C7F5 is from positions 148 to 198 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 8E7D9C7F5 is from positions 295 to 342 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 8E7D9C7F5 is from positions 70 to 114 of SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 8E7D9C7F5 is from position 160 to position 180 in SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 8E7D9C7F5 is from positions 277 to 303 in SEQ ID No. 114 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 20D1B6E9E5 is from positions 76 to 105 of SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 20D1B6E9E5 is 148th to 198th in SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 20D1B6E9E5 is from positions 295 to 339 of SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 20D1B6E9E5 is from positions 70 to 117 of SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 20D1B6E9E5 is from position 163 to position 183 in SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 20D1B6E9E5 is from positions 280 to 306 in SEQ ID No. 116 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 78C11D2D12 is from positions 76 to 105 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 78C11D2D12 is from positions 148 to 198 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 78C11D2D12 is position 295 to 315 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 78C11D2D12 is from position 70 to position 99 of SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 78C11D2D12 is from positions 145 to 165 in SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 78C11D2D12 is SEQ ID No. in the sequence listing. From 262th to 288th in 118.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 89A2G5E7 is from positions 76 to 105 of SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 89A2G5E7 is 148th to 195th in SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 89A2G5E7 is 292th to 327th in SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 89A2G5E7 is the 70th to 105th positions in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 89A2G5E7 is from positions 151 to 171 in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 89A2G5E7 is from positions 268 to 294 in SEQ ID No. 120 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 97E7B3F2 is from positions 76 to 105 of SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 97E7B3F2 is from positions 148 to 198 in SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 97E7B3F2 is from positions 295 to 321 of SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 97E7B3F2 is from positions 70 to 117 of SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 97E7B3F2 is from position 163 to position 183 in SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 97E7B3F2 is from positions 280 to 306 in SEQ ID No. 122 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 97A3C2H4 is from positions 76 to 105 of SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 97A3C2H4 is 148th to 198th in SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 97A3C2H4 is 295th to 327th in SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 97A3C2H4 is from positions 70 to 102 of SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 97A3C2H4 is 148th to 168th in SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 97A3C2H4 is from positions 265 to 291 in SEQ ID No. 124 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 67A2E1D10 is from positions 76 to 105 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 67A2E1D10 is from positions 148 to 198 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 67A2E1D10 is from positions 295 to 333 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 67A2E1D10 is from positions 70 to 102 of SEQ ID No. 126 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 67A2E1D10 is from positions 148 to 168 of SEQ ID No. 126 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 67A2E1D10 is position 265 to 291 of SEQ ID No. 126 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 111D10D6G3 is from positions 76 to 105 of SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 111D10D6G3 is 148th to 198th in SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 111D10D6G3 is from positions 295 to 309 of SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 111D10D6G3 is from positions 70 to 102 of SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 111D10D6G3 is 148th to 168th in SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 111D10D6G3 is from position 265 to position 291 in SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 93C6F10D3 is SEQ ID No. 129 of the Sequence Listing. 76th to 105th;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 93C6F10D3 is 148th to 198th in SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 93C6F10D3 is from positions 295 to 336 of SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 93C6F10D3 is from positions 70 to 102 of SEQ ID No. 130 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 93C6F10D3 is from positions 148 to 168 of SEQ ID No. 130 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 93C6F10D3 is from position 265 to position 291 in SEQ ID No. 130 of the Sequence Listing.
  • mouse-human chimeric BLyS antibody The antibody heavy chain variable region and light chain variable region sequences were obtained according to the sequencing results of the previous step. Production and preparation of mouse-human chimeric BLyS antibody Referring to step (3) of Example 2, ie, 1.
  • Preparation of recombinant vector directional cloning of the heavy chain variable region into a signal peptide and a human antibody heavy chain
  • An expression vector for the IgG1 constant region (wherein the expression vector was purchased from Invitrogen, the recombination step was performed by Shanghai Ruizhi Chemical Research Co., Ltd.), and the light chain variable region was directionally cloned into an expression vector comprising a signal peptide and a human antibody light chain kappa constant region.
  • the expression vector was purchased from Invitrogen, the recombination step was completed by Shanghai Ruizhi Chemical Research Co., Ltd.); 2. Cell transfection; 3. Antibody purification.
  • the obtained BLyS chimeric antibody was characterized (see Examples 3 to 5 for the method).
  • Each of the chimeric antibodies produced is named before the corresponding leader antibody clone number plus the first character "c".
  • the chimeric antibody c8E7D9C7F5 corresponds to the leader antibody clone number 8E7D9C7F5.
  • Table 21 shows that the chimeric antibody binds to the BLyS recombinant protein at the ELISA level.
  • the data in the table is the OD 450 nm value.
  • the binding and dissociation of the antibody to the antigen was detected by a Biacore instrument.
  • the results are shown in Table 22, indicating BLyS embedded
  • the antibody has a high affinity for hBLyS-ECD.
  • Table 23 shows that BLyS chimeric antibodies block BLyS protein and its receptor
  • the combination of BAFF R is the OD 450 nm value.
  • Table 24 demonstrates that BLyS chimeric antibodies can inhibit mouse B cell proliferation induced by hBLyS-ECD stimulation.
  • mice Female BALB/c mice (8-9 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were received at the SPF level after receiving, and the experiment was started after 1 week of adaptation.
  • the mice were injected intravenously with the whole human or human mouse chimeric BLyS monoclonal antibody prepared in Examples 2 and 6 on the first day and the third day (1 hour before the immunogen injection), and the clone numbers were 2-1G11 and L1D12, respectively.
  • the immunogen (hBLyS-ECD) prepared in Example 1 was subcutaneously injected daily from day 1 to day 4 for stimulation induction at a dose of 0.3 mg/kg. All animals were sacrificed on the fifth day, and the spleen weight of the mice, the proportion of B cells in the spleen cells, and the concentration of IgA in the serum were measured.
  • the human germline antibody heavy and light chain variable region templates that best match the non-CDR regions of the chimeric antibodies c8E7D9C7F5 or c97A3C2H4 described above were selected in the Germline database.
  • the sequence of the humanized BLyS antibody is selected from the human germline exon VH , JH , Vk and Jk sequences.
  • c8E7D9C7F5 template antibody heavy chain variable region of an antibody heavy chain human germline outer V H exon V H 1-18, outer exon J H J H -6, the template for the light chain variable region of human germline antibody light chain V K outer exon B3, J K exon J K -4.
  • Residues three dimensional structure of the murine antibody based on the embedded residues, and CDR regions have direct interaction of residues, and has an important influence on the V H and V L, conformation framework regions were back mutations to give Humanized antibody.
  • the amino acid sequence alignment of the heavy and light chain variable regions of the humanized BLyS antibody variant with the heavy and light chain variable regions of the chimeric antibody is shown in Table 26.
  • the heavy chain variable region sequences of the humanized BLyS antibody h8E7D9C7F5 variant are SEQ ID No. 140, SEQ ID No. 141, SEQ ID No. 142, SEQ ID No. 143, SEQ ID No. 144, SEQ ID, respectively.
  • No. 145, SEQ ID No. 146, the light chain variable region sequences are SEQ ID No. 147, SEQ ID No. 148, respectively.
  • the heavy chain variable region sequences of the humanized BLyS antibody h97A3C2H4 variant are SEQ ID No.
  • SEQ ID No. 150 SEQ ID No. 151
  • SEQ ID No. 152 respectively, and the light chain variable region sequences are respectively SEQ ID No. 153, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157.
  • the nucleotide sequences of the heavy and light chain variable regions of the humanized BLyS antibody variant are shown in Table 27.
  • the nucleotide sequence of the heavy chain variable region of the humanized BLyS antibody h8E7D9C7F5 variant is SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No. 161, SEQ ID No. 162, respectively.
  • SEQ ID No. 163, SEQ ID No. 164, the light chain variable region nucleotide sequence is SEQ ID No. 165, SEQ ID No. 166, respectively.
  • the heavy chain variable region nucleotide sequence of the humanized BLyS antibody h97A3C2H4 variant is SEQ ID No.
  • nucleotide sequence is SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173, SEQ ID No. 174, SEQ ID No. 175, respectively.
  • Antibody name Heavy chain variable region Light chain variable region h97A3C2H4-1 167 171 h97A3C2H4-2 167 172 h97A3C2H4-3 168 171 h97A3C2H4-4 168 172 h97A3C2H4-5 169 171 h97A3C2H4-6 169 172 h97A3C2H4-7 169 173 h97A3C2H4-8 169 174 h97A3C2H4-9 169 175 h97A3C2H4-10 170 173 h97A3C2H4-11 170 174 h97A3C2H4-12 170 175
  • the VH domain was directionally cloned into an expression vector containing the signal peptide and the human antibody heavy chain IgG1 constant region using a restriction site incorporated into the PCR product (the expression vector was purchased from Invitrogen, and the recombination step was performed by Shanghai Ruizhi Chemical Research Co., Ltd.
  • Table 28 shows that the humanized antibody binds to the BLyS recombinant protein at the ELISA level.
  • the data in the table is the OD 450 nm value.
  • Table 30 demonstrates that the BLyS humanized antibody is capable of blocking the binding of the BLyS protein to its receptor BAFF R.
  • the data in the table is the OD 450 nm value.
  • Table 31 demonstrates that BLyS humanized antibodies can inhibit mouse B cell proliferation induced by hBLyS-ECD stimulation.

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Abstract

一种BLyS抗体及其制备方法和应用，所述BLyS抗体包括BLyS抗体的重链可变区重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或BLyS抗体的轻链可变区轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，其氨基酸序列如序列表所示。所述BLyS抗体具有高亲和力，能明显在蛋白水平和细胞水平有效封闭BLyS蛋白，阻止BLyS蛋白与受体的结合。所述BLyS抗体缺乏与人APRIL等同类蛋白抗原的交叉反应，并且具有良好的生物活性，其能抑制人BLyS诱导的小鼠B细胞的增殖，因此可运用于预防或治疗与BLyS表达或功能异常相关的疾病的药物的制备中。

Description

一种BLyS抗体及其制备方法和应用
本申请要求申请日为2016年7月6日的中国专利申请CN201610527996.0的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于抗体领域，具体地涉及一种BLyS抗体及其制备方法和应用。
背景技术
自体免疫疾病(Autoimmune disease)为一种人体自身的免疫系统会攻击自己正常的器官、组织和细胞的疾病。它属于慢性病，一旦患上身体就会变得很虚弱，且无法治愈，只能控制。这导致患者需要承担高额的医疗费用并且生活质量急剧降低，从而对病人和他们的家庭甚至社会造成沉重的负担。大众比较熟悉的自体免疫疾病有系统性红斑狼疮(Systemic lupus erythematosus,SLE)或类风湿性关节炎(Rheumatoid arthritis,RA)等多种疾病。SLE可能影响各种器官，目前无法预测其何时发病，其自然病程多表现为病情的加重和缓解交替。SLE的全球平均患病率为每10万人中有12-39人，我国人群患病率为每10万人中有30-70人，仅次于黑人(100/10万)，位居全球第二位。SLE通常发生于年轻女性，有90％的患者是女性。RA是一个主要影响关节的长期持续性疾病，它通常导致关节发热、肿胀和疼痛，严重时会导致关节表面侵蚀及破坏，甚至造成肢体畸形。据统计，类风湿性关节炎的发病率为0.3％左右。近年来，我国乃至全球的自体免疫疾病发病率呈逐渐上升趋势。由于我国幅员辽阔、人口众多，因此按此发病率计算出的病人数量相当巨大。然而，目前仍然缺乏非常有效、副作用小、特异性强的干预治疗手段来早期阻断靶器官的损伤进而改善病人的预后。
研究表明，在自体免疫疾病和B细胞肿瘤的病人中能检测到B淋巴细胞刺激因子(B lymphocyte stimulator,BLyS)表达水平的提高。例如，在系统性红斑狼疮(SLE)的多组病人中都发现血清中BLyS的表达水平增加，并且这和自体免疫抗体的产生及疾病活动指数相关[参见Stohl et al.2003,Arthritis Rheum,48(12):3475；Petri et al.2008,Arthritis Rheum,58(8):2453]。在类风湿性关节炎(RA)病人的关节滑液(Synovial fluid)中也发现高水平的BLyS[参见Tan et al.2003,Arthritis Rheum,48(4):982]。BLyS的过表达和这些自体免疫疾病临床表现的相关性表明，调节BLyS的表达水平可以成为治疗这些疾病的新方法。
BLyS，也被称为BAFF、THANK、TALL-1、TNFSF13B或zTNF4，是肿瘤坏死因子(TNF)配体超家族的一员[参见Baker et al.2003,Arthritis Rheum,48(11):3253]。BLyS是由285个氨基酸组成的II型跨膜蛋白，有膜结合型和切割后具有152个氨基酸的可溶型两种形态。BLyS在单核细胞、巨噬细胞和树突细胞上都有表达并且在干扰素γ和白介素10刺激下表达水平上调。在体外，重组人BLyS可以通过与B细胞表面主要的受体结合，增强B细胞增殖和抗体分泌。在体内，重组人BLyS会导致小鼠的脾增生，这主要归因于成熟B细胞数目的增加。将BLyS注射到小鼠体内也会导致血清中抗体浓度的增加，以及针对T细胞依赖和非依赖抗原的体液免疫的增强。BLyS的过表达会产生表达水平异常高的抗体，从而导致SLE、RA及其它自体免疫疾病。
近几年来，单克隆抗体药物由于其具有特异性强、疗效好、副作用小等优点，被越来越广泛地应用于对肿瘤、自体免疫疾病等的治疗中。单克隆抗体药物的全球年销售额从1997年的3亿美元发展到2012年的663亿美元，已经成为生物医药产业中发展速度最快、盈利能力最强的领域之一，具有广阔的发展空间。目前，针对BLyS的单克隆抗体Belimumab作为半个世纪以来第一个获得FDA批准的治疗SLE的单克隆抗体，具有非常重要的意义。
尽管治疗SLE的单克隆抗体Belimumab还存在一些问题，但是与环磷酰胺和激素类等免疫抑制剂相比，单克隆抗体具有靶向性特点，副作用明显减少。预计到2022年，治疗SLE药物的销售额将达到39亿美元，市场潜力巨大。因此亟待获得全新的、更安全和更有效的针对BLyS的抗体来治疗SLE等自体免疫疾病。
发明内容
本发明所要解决的技术问题是为了克服目前缺少有效和安全的BLyS抗体的不足，提供一种亲和力高、特异性强的人源化或全人源的BLyS抗体及其制备方法和应用。本发明所述的BLyS抗体，与BLyS具有高度亲和力；能够抑制BLyS与其受体的结合；能够抑制人BLyS诱导的小鼠B细胞的增殖；并且缺乏与人APRIL等BLyS同族蛋白抗原的交叉反应，因此能够运用于制备治疗或预防自身免疫性疾病或肿瘤等与BLyS表达或功能异常相关的疾病的药物中。
本发明人采用噬菌体展示和杂交瘤技术，获得BLyS抗体的先导抗体。再通过对先导抗体的初步生产、纯化和鉴定，获得具备抗体亲和力高(亲和力KD＜5×10-9M)、能够有效封闭BLyS与受体的结合、与人ARRIL等同类蛋白抗原缺乏交叉反应等优异的生物活性的BLyS抗体。然后通过分子生物学方法测序获知BLyS抗体的重链可变区和BLyS 抗体的轻链可变区的氨基酸序列。
本发明提供一种分离的蛋白质，其包括BLyS抗体的互补决定区(CDR或CDRs)：重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，BLyS抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82、SEQ ID No.90或SEQ ID No.98所示；所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83、SEQ ID No.91所示或SEQ ID No.99；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84、SEQ ID No.92或SEQ ID No.100所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86、SEQ ID No.94或SEQ ID No.102所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87、SEQ ID No.95或SEQ ID No.103所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88、SEQ ID No.96或SEQ ID No.104所示；
或者，所述重链CDR1的氨基酸序列如与序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82、SEQ ID No.90或SEQ ID No.98所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列如与序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83、SEQ ID No.91所示或SEQ ID No.99所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列如与序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84、SEQ ID No.92或SEQ ID  No.100所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR1的氨基酸序列如与序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86、SEQ ID No.94或SEQ ID No.102所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列如与序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87、SEQ ID No.95或SEQ ID No.103所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列如与序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88、SEQ ID No.96或SEQ ID No.104所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.66所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.68所示；所述重链CDR1的 氨基酸序列如序列表SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.76所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.82所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.83所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.84所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.90所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.91所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.92所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.98所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.99所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.100所示；
所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.72所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.80所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.86所示，所述轻链CDR2的氨基酸序列如序列表SEQ  ID No.87所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.88所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.94所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.95所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.96所示；或者，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.102所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.103所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.104所示。较佳地，上述蛋白质包括由上述CDR区和构架区组成的BLyS抗体的重链可变区和/或BLyS抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65、SEQ ID No.73、SEQ ID No.81、SEQ ID No.89或SEQ ID No.97所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85、SEQ ID No.93或SEQ ID No.101所示。
本发明还提供一种分离的蛋白质，其包括BLyS抗体的重链可变区和/或BLyS抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65、SEQ ID No.73、SEQ ID No.81、SEQ ID No.89或SEQ ID No.97所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85、SEQ ID No.93或SEQ ID No.101所示。
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的氨基酸序列 如序列表SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.65所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.69所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.77所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.81所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.85所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.89所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.93所示；或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.97所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.101所示。
本领域中，抗体和抗原的结合区均含有一条轻链可变区和一条重链可变区，每一个可变区均含有CDR1、CDR2和CDR3三个结构域。
综上所述，上述氨基酸序列的编号如表1所示：
表1 BLyS抗体氨基酸序列编号
Figure PCTCN2017091839-appb-000001
其中，表1中的数字即为序列表中序列号，如2-1G11的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而2-1G11的重链蛋白可变区中CDR1的氨基酸序列如SEQ ID No.2所示。
本发明的分离的蛋白质还可以包括BLyS抗体的构架区(或称框架区或骨架区)， 所述构架区包括重链构架区和/或轻链构架区；较佳地，所述重链构架区为人或鼠抗体重链构架区，和/或，所述轻链构架区为人或鼠抗体轻链构架区。
更佳地，当本发明分离的蛋白质为人源化抗体或全人源抗体时，所述重链构架区为人抗体重链构架区，人抗体重链构架区残基可以包含种系DP4、DP7、DP8、DP9、DP10、DP14(VH1-18)、DP31、DP33、DP35(VH3-11)、DP45、DP46、DP47、DP48、DP49(VH3-30)、DP50、DP51(VH3-48)、DP53、DP54(VH3-7)、DP65、DP66、DP67、DP68和DP69，特别是这些种系的FR1、FR2、FR3；以及JH片段JH-1、JH-2、JH-3、JH-4、JH-4b、JH-5和JH-6，特别是这些种系的FR4编码的序列，或重链构架区的共有序列。所述轻链构架区为人抗体轻链构架区，人抗体轻链构架区残基可以包含种系O2、O12、DPK1(O18)、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、DPK12(A2)、DPK13、DPK15、DPK16、DPKI8、DPK19、DPK20、DPK21、DPK22、DPK23、DPK24(B3)、DPK25、DPK26(A10)和DPK 28，特别是这些种系的FR1、FR2、FR3；以及由Jk片段Jk1、Jk2、Jk3、Jk4和JK5，特别是这些种系的FR4编码的序列。此类构架区序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在"VBase"人种系序列数据库(http://www2.mrc-lmb.cam.ac.uk/vbase/)获得，以及在Kabat(E.A等人，1991Sequences of Proteins of Immunological Interest，第5版)中找到。在本发明人源化抗体的一较佳实施例中，重链构架区残基优选人种系抗体重链VH外显子的VH1-18、VH外显子的VH3-7或者JH外显子的JH-6，轻链构架区残基优选为人种系抗体轻链VK外显子的B3、JK外显子的JK-4或者VK外显子的A10。
较佳地，所述的蛋白质还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为小鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为小鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
其中，氨基酸序列如SEQ ID NO.25、33、41、49、57、65、73、81、89或者97所示的重链可变区以及序列如SEQ ID NO.29、37、45、53、61、69、77、85、93或者101所示的轻链可变区可与鼠源重链恒定区以及鼠源轻链恒定区构成鼠源化BlyS抗体、可与人源重链恒定区以及人源轻链恒定区构成BlyS嵌合抗体。
进一步地，将上述嵌合抗体中氨基酸序列如SEQ ID NO.25、33、41、49、57、65、73、81、89或者97所示的重链可变区中根据Kabat定义确定的序列如SEQ ID NO.26-28、34-36、42-44、50-52、58-60、66-68、74-76、82-84、90-92或者98-100所示的重链CDR 以及上述序列如SEQ ID NO.29、37、45、53、61、69、77、85、93或者101的轻链可变区中根据Kabat定义确定的序列如SEQ ID NO.30-32、38-40、46-48、54-56、62-64、70-72、78-80、86-88、94-96或者102-104所示的轻链CDR分别移植到所选人种系模板中，替换人种系模板的CDR区，即得人源化的抗体；所述种系模板中的轻链构架区和重链构架区如上所述，优选地分别选自人种系抗体重链VH外显子的VH1-18、VH外显子的VH3-7和JH外显子的JH-6，以及人种系抗体轻链VK外显子的B3、JK外显子的JK-4和VK外显子的A10。选择性地，为了保证抗体活性，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VH和VL构象有重要影响的构架区残基进行回复突变。
较佳地，上述CDR区被移植到所选人种系模板中且构架区残基经过回复突变后的重链可变区的氨基酸序列优选如SEQ ID NO.140、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151或SEQ ID NO.152所示；轻链可变区的氨基酸序列优选如SEQ ID No.147、SEQ ID No.148、SEQ ID No.153、SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、或SEQ ID No.157所示。
序列如SEQ ID NO.1、9或者17所示的重链可变区以及序列如SEQ ID NO.5、13或者21所示的轻链可变区可与人源抗体重链恒定区以及人源抗体轻链恒定区构成全人源BlyS抗体。
所述的蛋白质是指抗体蛋白质，较佳地，其为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其 包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)2。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述蛋白质的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述蛋白质。
本发明还提供一种核酸，其编码上述的蛋白质。
较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.105、SEQ ID No.107、SEQ ID No.109、SEQ ID No.111、SEQ ID No.113、SEQ ID No.115、SEQ ID No.117、SEQ ID No.119、SEQ ID No.121、SEQ ID No.123、SEQ ID No.125、SEQ ID No.127或SEQ ID No.129所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106、SEQ ID No.108、SEQ ID No.110、SEQ ID No.112、SEQ ID No.114、SEQ ID No.116、SEQ ID No.118、SEQ ID No.120、SEQ ID No.122、SEQ ID No.124、SEQ ID No.126、SEQ ID No.128或SEQ ID No.130所示。
更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.107所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.108所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.109所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.110所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.112所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.114所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.116所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；编码所述重 链可变区的核酸的核苷酸序列如序列表SEQ ID No.121所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.122所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.123所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.124所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.125所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.126所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.127所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.128所示；或者，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.129所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.130所示。
综上所述，上述核苷酸序列的编号如表2所示：
表2 BLyS抗体基因核苷酸序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
2-1G11 105 106
L9G7 107 108
L1D12 109 110
35E6F7C3 111 112
8E7D9C7F5 113 114
20D1B6E9E5 115 116
78C11D2D12 117 118
89A2G5E7 119 120
97E7B3F2 121 122
97A3C2H4 123 124
67A2E1D10 125 126
111D10D6G3 127 128
93C6F10D3 129 130
其中，表2中的数字即为序列表中序列号，如编码2-1G11的重链蛋白可变区的氨基酸序列的核苷酸序列为SEQ ID No.105，而编码2-1G11的轻链蛋白可变区的氨基酸序列的核苷酸序列为SEQ ID No.106。
编码2-1G11的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.105中的第76位至第105位；
编码2-1G11的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.105中的 第148位至第198位；
编码2-1G11的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.105中的第295位至第342位；
编码2-1G11的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.106中的第70位至第102位；
编码2-1G11的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.106中的第148位至第168位；
编码2-1G11的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.106中的第265位至第291位；
编码L9G7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.107中的第76位至第105位；
编码L9G7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.107中的第148位至第198位；
编码L9G7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.107中的第295位至第342位；
编码L9G7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.108中的第70位至第102位；
编码L9G7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.108中的第148位至第168位；
编码L9G7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.108中的第265位至第291位；
编码L1D12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.109中的第76位至第105位；
编码L1D12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.109中的第148位至第195位；
编码L1D12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.109中的第292位至第330位；
编码L1D12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.110中的第67位至第99位；
编码L1D12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.110中的第145位至第165位；
编码L1D12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.110中的第262位至第297位；
编码35E6F7C3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.111中的第76位至第105位；
编码35E6F7C3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.111中的第148位至第198位；
编码35E6F7C3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.111中的第295位至第321位；
编码35E6F7C3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.112中的第70位至第99位；
编码35E6F7C3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.112中的第145位至第165位；
编码35E6F7C3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.112中的第262位至第288位；
编码8E7D9C7F5的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.113中的第76位至第105位；
编码8E7D9C7F5的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.113中的第148位至第198位；
编码8E7D9C7F5的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.113中的第295位至第342位；
编码8E7D9C7F5的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.114中的第70位至第114位；
编码8E7D9C7F5的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.114中的第160位至第180位；
编码8E7D9C7F5的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.114中的第277位至第303位；
编码20D1B6E9E5的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.115中的第76位至第105位；
编码20D1B6E9E5的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.115中的第148位至第198位；
编码20D1B6E9E5的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No. 115中的第295位至第339位；
编码20D1B6E9E5的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.116中的第70位至第117位；
编码20D1B6E9E5的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.116中的第163位至第183位；
编码20D1B6E9E5的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.116中的第280位至第306位。
编码78C11D2D12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.117中的第76位至第105位；
编码78C11D2D12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.117中的第148位至第198位；
编码78C11D2D12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.117中的第295位至第315位；
编码78C11D2D12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.118中的第70位至第99位；
编码78C11D2D12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.118中的第145位至第165位；
编码78C11D2D12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.118中的第262位至第288位；
编码89A2G5E7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.119中的第76位至第105位；
编码89A2G5E7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.119中的第148位至第195位；
编码89A2G5E7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.119中的第292位至第327位；
编码89A2G5E7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.120中的第70位至第105位；
编码89A2G5E7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.120中的第151位至第171位；
编码89A2G5E7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.120中的第268位至第294位。
编码97E7B3F2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.121中的第76位至第105位；
编码97E7B3F2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.121中的第148位至第198位；
编码97E7B3F2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.121中的第295位至第321位；
编码97E7B3F2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.122中的第70位至第117位；
编码97E7B3F2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.122中的第163位至第183位；
编码97E7B3F2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.122中的第280位至第306位。
编码97A3C2H4的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.123中的第76位至第105位；
编码97A3C2H4的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.123中的第148位至第198位；
编码97A3C2H4的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.123中的第295位至第327位；
编码97A3C2H4的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.124中的第70位至第102位；
编码97A3C2H4的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.124中的第148位至第168位；
编码97A3C2H4的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.124中的第265位至第291位；
编码67A2E1D10的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.125中的第76位至第105位；
编码67A2E1D10的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.125中的第148位至第198位；
编码67A2E1D10的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.125中的第295位至第333位；
编码67A2E1D10的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.126 中的第70位至第102位；
编码67A2E1D10的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.126中的第148位至第168位；
编码67A2E1D10的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.126中的第265位至第291位。
编码111D10D6G3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.127中的第76位至第105位；
编码111D10D6G3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.127中的第148位至第198位；
编码111D10D6G3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.127中的第295位至第309位；
编码111D10D6G3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.128中的第70位至第102位；
编码111D10D6G3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.128中的第148位至第168位；
编码111D10D6G3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.128中的第265位至第291位；
编码93C6F10D3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.129中的第76位至第105位；
编码93C6F10D3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.129中的第148位至第198位；
编码93C6F10D3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.129中的第295位至第336位；
编码93C6F10D3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.130中的第70位至第102位；
编码93C6F10D3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.130中的第148位至第168位；
编码93C6F10D3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.130中的第265位至第291位。
在本发明较佳实施例中，编码上述经人构架区改造的本发明人源化抗体的所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO.158、SEQ ID NO.159、SEQ ID NO. 160、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.163、SEQ ID NO.164、SEQ ID NO.167、SEQ ID NO.168、SEQ ID NO.169或SEQ ID NO.170所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165、SEQ ID No.166、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173、SEQ ID No.174、或SEQ ID No.175所示。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明还提供一种BLyS抗体的制备方法，其包括如下步骤：培养上述的重组表达转化体，从培养物中获得BLyS抗体。
本发明还提供一种检测过表达BLyS蛋白的细胞的方法，包括如下的步骤：上述的蛋白质与待检样品在体外接触，检测上述的蛋白质与所述待检样品的结合即可。
所述的过表达的含义为本领域常规，指BLyS蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表达和功能活性提高(如在底物 的酶水解作用增加的情况下)。
本发明中，上述结合的检测方式是本领域常规的检测方式，较佳地为流式细胞实验(FACS)检测。
本发明提供一种检测过表达BLyS蛋白的细胞的组合物，其包括上述的蛋白质作为活性成分。较佳地，其还包括上述的蛋白质的功能片段组成的化合物作为活性成分。
本发明提供上述蛋白质在制备药物中的应用。
较佳地，所述的药物是用于预防或治疗与BLyS表达或功能异常相关的疾病的药物。
本发明中，所述与BLyS表达或功能异常相关的疾病是本领域常规的与BLyS表达或功能异常相关的疾病。较佳地，为自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病。本发明中，所述自体免疫疾病为本领域常规的自体免疫疾病，较佳地为红斑狼疮(SLE)、类风湿性关节炎(RA)、干燥综合症(SS)、多发性硬化(MS)、重症肌无力(MG)、慢性甲状腺炎或免疫缺陷综合症。本发明中，所述炎症性疾病为本领域常规的炎症性疾病，较佳地为哮喘或过敏性疾病。本发明中，所述感染性疾病为本领域常规的感染性疾病。较佳地，为获得性免疫缺陷综合症(AIDS)。本发明中，所述增殖性疾病为本领域常规的增殖性疾病，较佳地为白血病、肿瘤或淋巴瘤。
本发明还提供一种药物组合物，其活性成分包括上述的蛋白质。
较佳地，所述的药物组合物是用于预防或治疗与BLyS表达或功能异常相关的疾病的药物组合物。
本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
本发明提供上述蛋白质在制备预防或治疗与BLyS表达或功能异常相关的疾病的药物中的应用。较佳地，所述与BLyS表达或功能异常相关的疾病为自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病。
本发明提供上述药物组合物在制备预防或治疗BLyS表达或功能异常相关的疾病的药物中的应用。较佳地，所述与BLyS表达或功能异常相关的疾病为自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明所述的蛋白质是一种人源化或全人源BLyS抗体，其与BLyS蛋白具有高度亲和力(亲和力KD＜5*10-9M)，能够在蛋白水平和细胞水平有效封闭BLyS蛋白，阻止BLyS蛋白与受体的结合。所述的BLyS抗体缺乏与人APRIL等同类蛋白抗原的交叉反应。B细胞增殖实验证明，所述的BLyS抗体具有良好的生物活性，其能抑制人BLyS诱导的小鼠B细胞的增殖。小鼠体内检测证明，所述的BLyS抗体能够降低由BLyS蛋白引起的小鼠脾细胞中B细胞比例的增加。因此所述的BLyS抗体单独或与其它药物联合能够用于检测、诊断、治疗或筛查与BLyS表达或功能异常相关的疾病，如自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病。
附图说明
以下结合附图说明本发明的特征和有益效果。
图1为纯化的hBLyS-ECD的生物活性检测的结果图。其中，RLU表示相对光单位。
图2A和图2B为流式细胞分析方法检测CHO-K1重组细胞系BLyS蛋白的表达水平的结果图。其中，2C4表示克隆号；4C4表示克隆号；抗体指BLyS抗体(购自eBioscience)；阴性对照指同型抗体对照。
图3为ELISA检测免疫原免疫后BALB/c和SJL小鼠血清抗体效价的结果图。
图4为酶联免疫吸附检测生物素化的hBLyS-ECD的活性的结果图。
图5A和图5B为酶联免疫吸附实验中BLyS纯化抗体与生物素化的hBLyS-ECD的反应活性的结果图。
图6A和图6B为酶联免疫吸附实验检测BLyS纯化抗体与BLyS同家族APRIL蛋白的反应的结果图。
图7为流式细胞分析实验检测BLyS纯化抗体与人BLyS重组细胞反应活性的结果 图。
图8为流式细胞分析实验检测BLyS纯化抗体与猴BLyS重组细胞反应活性的结果图。
图9A和图9B为BLyS纯化抗体封闭BLyS受体BAFF R与生物素化的hBLyS-ECD的结合活性的结果图。
图10为BLyS纯化抗体对由hBLyS-ECD刺激的小鼠B细胞增殖的抑制作用的结果图。
图11为BLyS纯化抗体对由鼠BLyS刺激的小鼠B细胞增殖的抑制作用的结果图。
图12为酶联免疫吸附实验中BLyS嵌合抗体与生物素化的hBLyS-ECD的反应活性的结果图。
图13为BLyS嵌合抗体封闭BLyS受体BAFF R与生物素化的hBLyS-ECD的结合活性的结果图。
图14为BLyS嵌合抗体对由hBLyS-ECD刺激的小鼠B细胞增殖的抑制作用的结果图。
图15A～图15C为BLyS嵌合抗体及全人源抗体对hBLyS-ECD刺激后的小鼠B细胞在脾细胞中所占比例的影响的结果图。
图16为酶联免疫吸附实验中BLyS人源化抗体与生物素化的hBLyS-ECD的反应活性的结果图。
图17为BLyS人源化抗体封闭BLyS受体BAFF R与生物素化的hBLyS-ECD的结合活性的结果图。
图18为BLyS人源化抗体对由hBLyS-ECD刺激的小鼠B细胞增殖的抑制作用的结果图。
图19A～图19B为BLyS人源化抗体对hBLyS-ECD刺激后的小鼠B细胞在脾细胞中所占比例以及血清中IgA水平的影响的结果图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例中所述的“室温”是指进行试验的操作间的温度，一般为15～30℃。
实施例1杂交瘤技术制备BLyS抗体
(一)、免疫原的制备
将含有编码人源BLyS蛋白胞外区(BLyS-ECD)中Ala134-Leu285的氨基酸序列的核苷酸序列(如序列表SEQ ID No.131所示)克隆到带有His标签的pCPC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒，具体方法参见[Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)]。对HEK293细胞(购自Invitrogen)进行瞬时转染(PEI，Polysciences)并使用FreeStyle TM 293(购自Invitrogen)在37℃下进行扩大培养。7天后收集细胞培养液，离心去除细胞成分，得含人源BLyS蛋白胞外区的培养上清液。将培养上清液上样到Ni亲和层析柱(购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用含有5％(v/v)蔗糖和0.01％(v/v)Tween-80的磷酸缓冲液(pH7.4)清洗Ni亲和层析柱，直到紫外吸收值回到基线，然后用0-500mM的咪唑进行梯度洗脱。收集从Ni亲和层析柱上洗脱下来的带His标签的人源BLyS蛋白胞外区，使用含有5％(v/v)蔗糖和0.01％(v/v)Tween-80的磷酸缓冲液(pH7.4)在超滤管进行4℃下透析和浓缩蛋白。透析后的蛋白经0.22μm无菌过滤后分装于-80℃保存，获得纯化的人源BLyS蛋白胞外区，作为免疫原(即hBLyS-ECD)。该免疫原在使用前进行一系列质控检测，如检测其蛋白浓度、纯度、分子量、生物活性等。
其中，免疫原的生物活性采用B细胞增殖实验检测(方法参见实施例5)。结果如图1和表3所示，该免疫原可以刺激小鼠B细胞的增殖。
表3免疫原的生物活性检测
Figure PCTCN2017091839-appb-000002
(二)、表达人或猴BLyS的稳转细胞株的构建
将人或猴的BLyS(即hBLyS或cyno BLyS)的氨基酸序列(NCBI中的基因登陆号分别为Q9Y275、EHH58704.1)的第132位从精氨酸突变为组氨酸，使构建的稳转细胞株表达完整的人或猴的BLyS而不进行切割。将突变后的人或猴的BLyS全长氨基酸序列的核苷酸序列(如序列表SEQ ID No.132和SEQ ID No.133所示)克隆到pLVX载体(购自Clontech)中，由上海吉玛制药技术有限公司进行慢病毒包装，随后感染CHO-K1细胞系(购自Invitrogen)，得转染后的细胞。72个小时后，用已知的BLyS抗体(购自 eBioscience)经流式细胞分析法进行检测[参见Manetta J et al.,2014,J Inflamm Res,20(7):121-131]。当确定转染后的细胞开始表达人或猴的BLyS，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃、5％(v/v)CO2条件下培养，大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆再用已知的BLyS抗体(购自eBioscience)经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得表达人或猴BLyS的稳转细胞株。具体选择结果如表4和图2A～图2B所示，表4中阳性细胞(％)指阳性细胞占总细胞数目的百分比。表4说明，已经制得一系列BLyS阳性表达的CHO-K1细胞系。
表4表达人或猴BLyS蛋白的CHO-K1细胞FACS筛选检测结果
Figure PCTCN2017091839-appb-000003
(三)、杂交瘤细胞的制备和抗体筛选
A、采用6～8周龄雌性BALB/c和SJL小鼠(购自上海斯莱克实验动物有限责任公司)，小鼠在SPF条件下饲养。初次免疫时，将步骤(一)获得的免疫原(即hBLyS-ECD)用 弗氏完全佐剂乳化后腹腔注射0.2mL，即每只小鼠注射100μg免疫原。加强免疫时，免疫原用弗氏不完全佐剂乳化后腹腔注射0.2mL，即每只小鼠注射50μg免疫原。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA检测血清中免疫原的抗体效价和特异性，结果如图3和表5所示。表5说明，经免疫原免疫后的小鼠的血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一百万左右。其中空白对照为1％(w/w)BSA，其中批次指第二次加强免疫后第七天的小鼠血清，表中的数据为OD450nm值。
表5 ELISA检测免疫原免疫后BALB/c和SJL小鼠血清抗体效价
Figure PCTCN2017091839-appb-000004
B、采用6～8周龄雌性BALB/c和SJL小鼠(购自上海斯莱克实验动物有限责任公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原(hBLyS-ECD)、寡核苷酸(CpG)和GERBU佐剂混合后小鼠跗关节注射25μL，即每只小鼠注射10μg免疫原。加强免疫时，免疫原和GERBU佐剂混合后跗关节注射25μL，即每只小鼠注射5μg免疫原。初次免疫与加强免疫之间间隔3-4天。第二次加强免疫1周后采血，用ELISA检测血清中免疫原的抗体效价和特异性。在第二次加强免疫后，ELISA检测血清抗体效价达到1:10000以上。
步骤A和B完成前，向每只所选择的、最后一次免疫的小鼠的腹腔或者跗关节分别注射50μg或5μg免疫原，5天后处死小鼠，收集脾细胞。加入NH4OH至终浓度1％(w/w)，裂解脾细胞中参杂的红细胞，获得脾细胞悬液。用DMEM基础培养基(购自invitrogen)1000转每分钟离心清洗细胞3次，然后按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％(w/w)胎牛血清和1╳HAT的DMEM培养基中。然后按1╳105个/200μL每孔加入到96孔细胞培养板中，放入5％(v/v)CO2、37℃培养箱中。14天后用间接ELISA筛选细胞融合板上清，将ELISA中OD450nm>1.0的阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清的DMEM在37℃、5％(v/v)CO2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析。其中，用ELISA确定对hBLyS-ECD的结合活性(结合活 性的检测方法请分别参见实施例3A和实施例3B)，配体受体结合实验确定抗体样品对BLyS受体的封闭活性(封闭活性的检测方法请分别参见实施例4)。
根据24孔板筛选结果，挑选ELISA实验中OD450nm>1.0和配体受体结合实验中杂交瘤细胞培养上清对BLyS受体的封闭抑制率达到60％的杂交瘤细胞为符合条件的阳性克隆，选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自invitrogen)37℃、5％(v/v)CO2条件下培养。亚克隆后10天用ELISA进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用受体配体结合实验评估生物活性。其中，评估标准为ELISA实验中OD450nm>1.0，且配体受体结合实验中杂交瘤细胞培养上清对BLyS受体的封闭抑制率达到60％。
根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(w/w)FBS的DMEM培养基中(购自invitrogen)在37℃、5％(v/v)CO2条件下将该最优的克隆进行扩大培养，液氮冻存即得最优的杂交瘤细胞，并可用于后续的抗体生产和纯化。
(四)、先导抗体的生产和纯化
由于杂交瘤细胞产生的抗体浓度较低，仅约1～10μg/mL且所得的抗体浓度变化较大；此外，培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1～5mg)的抗体生产纯化。
将步骤(三)所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入500mL生产培养基，接种细胞密度为1.0╳105个/mL。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45μm的滤膜过滤至培养上清液澄清。澄清的培养上清液可马上进行纯化或置于-30℃冻存。
用2mL蛋白G柱(购自GE Healthcare)纯化300mL杂交瘤细胞的澄清的培养上清液中的单克隆抗体。蛋白G柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，然后将澄清的培养上清液上样到蛋白G柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白G柱，平衡缓冲液的体积为蛋白G柱的柱床体积的4倍。用洗脱液(0.1M甘氨盐酸缓冲液，pH2.5)洗脱结合在蛋白G柱上的单克隆抗体，用紫外检测器监测洗脱情况(A280紫外吸收峰)。收集洗脱的单克隆抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH。然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的单克隆抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的BLyS抗体作为先导抗体。
将纯化的BLyS抗体进行蛋白浓度(A280/1.4)、纯度、内毒(Lonza试剂盒)等检测分析，结果如表6所示。结果发现，先导抗体的内毒素浓度在1.0EU/mg以内。
表6纯化的BLyS抗体检测分析
Figure PCTCN2017091839-appb-000005
实施例2噬菌体展示技术制备BLyS抗体
(一)、BLyS蛋白的生物素化
将Biotin-X-X-NHS(购自Sigma Aldrich)和实施例1制备的免疫原(hBLyS-ECD)按摩尔比7:1的比例混合，室温放置30分钟后，加入终浓度为50mM的1M NH4Cl终止反应。然后用PBS磷酸缓冲液透析过夜来去除游离的生物素，得生物素化的免疫原(即生物素化的hBLyS-ECD)。用BCA蛋白浓度测定试剂盒(购自Pierce)测定生物素化的hBLyS-ECD的浓度。
生物素化的hBLyS-ECD的活性采用ELISA方法测定。将BLyS抗体(购自GSK)用PBS稀释至1μg/mL，以50μL/孔加入ELISA微孔板，4℃孵育过夜；用ELISA封闭液[含1％(w/v)BSA和0.05％(v/v)Tween-20的PBS磷酸缓冲液，pH7.4]37℃封闭2小时后，再加入梯度稀释的生物素化的hBLyS-ECD，37℃温育1小时。加入链霉亲和素标记的辣根过氧化物酶(购自Sigma，商品号S5512)，室温孵育30分钟。加入100μL/孔TMB显色液，室温孵育15分钟后，加入5μL 1N盐酸终止显色反应，用ELISA读板机读取OD450nm读数。结果如图4和表7所示，结果说明，生物素化的hBLyS-ECD可以结合BLyS抗体。
表7 ELISA检测生物素化的免疫原与BLyS抗体的结合
Figure PCTCN2017091839-appb-000006
(二)、噬菌体展示技术筛选BLyS抗体
利用天然人的单链抗体(ScFv)噬菌体展示库(由上海睿智化学研究有限公司构建)筛选先导抗体。通过四轮生物淘选得到与BLyS结合的抗体。具体过程如下：
将链霉亲和素用PBS稀释至12.5μg/mL，以1mL每管加入到免疫管中，4℃孵育过夜。用PBS洗涤免疫管3次后，在一半的免疫管中，每管加入50μg的步骤(一)制得的生物素化的hBLyS-ECD，室温振荡1小时。PBS缓冲液洗涤3次之后，免疫管用10mL2％(w/v)的封闭液[含有2％(w/v)脱脂奶粉的PBS缓冲液]室温封闭2小时。同时，在另一半免疫管中，加入1mL噬菌体ScFv抗体库和封闭液，室温振荡2小时。并且设置对照管，加入同溶液体积的封闭液。将加入生物素化的hBLyS-ECD的免疫管和对照管中的封闭液倒掉，加入已经封闭好的噬菌体ScFv抗体库，室温振荡2小时。免疫管和对照管先用PBST[含有0.1％(v/v)Tween-20的PBS缓冲液]洗涤5次，再用PBS缓冲液洗涤5次。其中，洗涤次数每一轮多增5次。洗涤后，每管加入1mL的10μg/mL胰酶，37℃孵育30分钟来洗脱与生物素化的hBLyS-ECD结合的噬菌体。将1mL的胰酶溶液加到4mL处于对数生长期的大肠杆菌TG1(购自LUCIGEN)中，37℃孵育30分钟，得TG1的培养液。将TG1的培养液梯度稀释，涂布平板，37℃培养过夜。计算所得的与生物素化的hBLyS-ECD结合的免疫管和对照管的克隆数，并挑选20～30个克隆测序。
同时，将平板上的克隆用2YT培养基(2YT培养基的配制方法为：将10g酵母提取物、16g胰蛋白胨和5g NaCl加入1L水中，再用NaOH调pH至7.0，高压灭菌)洗涤、收集，并接种到新鲜培养基中，37℃培养至对数期。加入辅助噬菌体M13KO7(购自NEB，货号N0315S)，混匀，37℃静置30分钟。然后37℃振荡培养30分钟，4000rpm离心10分钟后收集细胞，加入新鲜培养基，30℃振荡培养4小时。4000rpm离心30分钟，收集上清，加入上清体积的1/4体积的含有5×PEG的2.5M的NaCl溶液，放置冰上过夜。4000rpm，4℃离心30分钟，收集噬菌体沉淀，溶解在PBS缓冲液中。10000rpm离心10分钟去除残留的细胞碎片，收集上清用于下一轮的生物淘选。
用ELISA方法确定筛选得到的scFv抗体具有与BLyS蛋白的结合活性。将OD450nm>1.0的克隆挑选出来进行测序，得到具有不同HCDR3序列的克隆。然后再通过FACS和配体受体结合实验，选择FACS实验中MFI值>30并且在配体受体结合实验中细 胞裂解上清对BLyS受体的封闭抑制率达到60％的克隆作为符合条件的阳性克隆。
(三)、抗体的生产和纯化
根据阳性克隆的测序结果(具体参见下述实施例7)，设计引物(具体引物序列如表8所示)通过PCR方法分别扩增轻链和重链可变区。配置50μL反应体系，包括0.5μL含有转染阳性克隆大肠杆菌TG1中提取的质粒)、每种引物10pmol、0.5μL DNA聚合酶以及相配的缓冲体系。设置PCR程序，预变性95℃2分钟，变性95℃15秒，退火55℃30秒，延伸68℃45秒，30个循环后再额外于68℃延伸10分钟，得PCR产物。其中PCR所用的DNA聚合酶，购自Invitrogen，货号12344；缓冲体系为该DNA聚合酶配套购买使用的缓冲体系。取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2017091839-appb-000007
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：插入片段3μL，酶切过的表达载体0.5μL，重组酶Exnase 0.5μL，缓冲液2μL，反应体系10μL，于37℃反应半小时得连接产物，即构建好的重组载体。其中，重组酶购自Vazyme，货号C112-01/02；缓冲液为该重组酶配套购买使用的重组酶；将重链可变区定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤由上海睿智化学研究有限公司完成)，将轻链可变区定向克隆到包含信号肽和人源抗体轻链kappa或lambda(仅针对下述L1D12抗体的轻链)恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤由上海睿智化学研究有限公司完成)中。将10μL连接产物加入50μL的感受态细胞(Ecos101competent cells，购自Yeastern，货号FYE607)中，冰浴30分钟。再于42℃水浴热激1分钟，放回冰上2分钟后加入500μL无抗生素的2YT培养基，于37℃摇床上以200RPM的速度复苏45分钟，取出200μL涂布于含100μg/mL氨苄青霉素的LB固体培养基上于37℃孵箱过夜培养。次日，使用表达载体上引物pTT-EF1a-F和pSV40(其核苷酸序列分别为序列表SEQ ID No.134～135所示)，配置30μL PCR体系，进行菌落PCR。菌落PCR的体系为：引物各1μL，10μL的PCR预混液(购自Novoprotein)，用水补足到20μL。用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100μg/mL氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat,“Sequences of Proteins of Immunological Interest,”National Institutes of Health,Bethesda,Md.(1991)]。
表8阳性克隆的引物序列编号
Figure PCTCN2017091839-appb-000008
Figure PCTCN2017091839-appb-000009
经过菌落PCR验证，将上述序列正确的重组抗体轻、重链的表达载体瞬时转染到FreeStyleTM 293-F细胞(购自Invitrogen)中来生产抗体。在转染的时候，293-F细胞的密度应为1-1.5×106个/mL，并且100mL细胞需要100μg上述已构建好的载体DNA(其中，重组轻链载体和重链载体的质量比为3:2)和200μg的转染试剂聚乙烯亚胺(PEI)。将载体DNA和PEI分别加入到培养基中，室温静置5分钟，0.22μm滤膜过滤后，将载体DNA和PEI混合得混合物，室温静置15分钟。然后将上述混合物缓慢地加入到细胞中，在37℃、8％(v/v)CO2培养箱中以120rpm的转速培养。转染后的第二天，加入0.5％(v/v)蛋白胨到细胞的培养液中。6-7天后，3500g离心细胞的培养液30分钟，收集上清液，0.22μm滤器过滤。
200mL澄清的上清液中的单克隆抗体用1mL蛋白A柱(购自GE Healthcare)纯化。蛋白A柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，然后将上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白A柱，平衡缓冲液的体积为蛋白A柱的柱床体积的5倍。用洗脱液(0.1M甘氨盐酸缓冲液，pH3.0)洗脱结合在蛋白A柱上的BLyS抗体，用紫外检测器监测洗脱情况(A280紫外吸收峰)。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的BLyS抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的BLyS抗体作为先导抗体。
将先导抗体进行蛋白浓度(A280/1.4)、纯度、内毒(Lonza试剂盒)等检测分析。结果如表9所示，结果显示，先导抗体内毒素浓度在1.0EU/mg以内。
表9纯化的BLyS抗体检测分析
Figure PCTCN2017091839-appb-000010
实施例3先导抗体的检定
A、酶联免疫吸附实验(ELISA)检测抗体与BLyS蛋白的结合
将实施例1和2所得的先导抗体进行与人BLyS蛋白和BLyS蛋白所在家族的人APRIL蛋白分别进行交叉反应。
先将链霉亲和素用PBS稀释到终浓度1.0μg/mL，然后以50μL每孔加到96孔ELISA板，用塑料膜封好4℃孵育过夜。第二天用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次，加入封闭液[含有0.05％(v/v)Tween20和1％(w/w)BSA的PBS缓冲液]，37℃封闭2小时。倒掉封闭液，将实施例2获得的生物素化的hBLyS-ECD用PBS稀释到终浓度50ng/mL，然后以50μL每孔加到96孔ELISA板，37℃孵育1小时。用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次后，每孔加入实施例1和2所得的纯化的先导抗体100μL。37℃孵育1小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育1小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。每孔加入100μL TMB底物，室温孵育5分钟后，每孔加入100μL终止液(1.0N HCl)。用ELISA读板机(SpectraMax M5e，购自Molecular Device)读取A450nm数值，结果如图5A～图5B和表10所示，表10说明，纯化后的抗体与BLyS重组蛋白在ELISA水平结合。其中表中的数据为OD450nm值。
表10 ELISA检测BLyS纯化抗体与生物素化的hBLyS-ECD的结合反应
Figure PCTCN2017091839-appb-000011
检测BLyS抗体是否与人APRIL蛋白有交叉反应时，先将APRIL蛋白(购自R&D Systems)用PBS稀释到终浓度1.0μg/mL，然后以50μL每孔加到96孔ELISA板，用塑料膜封好4℃孵育过夜。第二天用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次，加入封闭液[含有0.05％(v/v)Tween20和1％(w/w)BSA的PBS缓冲液]，37℃封闭2小时。倒掉封闭液，加入实施例1和2所得的先导抗体100μL每孔。37℃孵育1 小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育1小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。每孔加入100μL TMB底物，室温孵育5分钟后，每孔加入100μL终止液(1.0N HCl)。用ELISA读板机(SpectraMax M5e，购自Molecular Device)读取A450nm数值，结果如图6A～图6B和表11所示，表11说明，先导抗体与BLyS蛋白同家族的APRIL重组蛋白在ELISA水平不结合。其中IgG对照为人IgG，表中的数据为OD450nm值。
表11-1 ELISA检测BLyS抗体与APRIL蛋白的结合反应
Figure PCTCN2017091839-appb-000012
表11-2 ELISA检测BLyS抗体与APRIL蛋白的结合反应
Figure PCTCN2017091839-appb-000013
B、流式细胞实验(FACS)检测抗体与BLyS表达细胞的结合
将实施例1步骤(二)获得的CHO-K1hBLyS稳定细胞株和CHO-K1cynoBLyS稳 定细胞株在T-175细胞培养瓶中扩大培养至90％汇合度。吸尽培养基，用PBS缓冲液(购自Invitrogen)洗涤1次，然后用细胞解离液(TrypLETM Express Enzyme，购自Life technology公司)处理和收集细胞。具体步骤为：用PBS缓冲液洗涤细胞1次，进行细胞计数后将细胞用PBS缓冲液稀释至2╳106个细胞每毫升，加入2％(w/w)胎牛血清封闭液，冰上孵育30分钟。按每孔200μL加入到96孔FACS反应板中，4℃离心2000rpm后，每孔加入100μL实施例1和2所得的先导抗体，冰上孵育1小时。用FACS缓冲液[含有2％(w/w)FBS的PBS缓冲液]离心洗涤2次，加入每孔100μL荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次。用200μL FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图7～8和表12～13所示，表12～13说明，先导抗体可结合细胞表面的人BLyS蛋白，且先导抗体和猴BLyS存在交叉反应。其中表中的数据为MFI所测细胞群的平均荧光强度值。
表12流式细胞分析实验检测BLyS抗体与人BLyS重组细胞(CHO-K1hBLyS)的结合反应
Figure PCTCN2017091839-appb-000014
表13流式细胞分析实验检测BLyS抗体与猴BLyS重组细胞(CHO-K1cynoBLyS)的结合反应
Figure PCTCN2017091839-appb-000015
C.BLyS抗体亲和常数的测定
使用Biacore X100仪器(购自GE Healthcare)进行亲和常数的测定。具体操作和方法根据仪器说明书和厂家提供的详细方法。具体为：用CM5芯片(Sensor Chip CM5，购自GE Healthcare)进行亲和力测定。首先将CM5芯片依次用50mM的NaOH和按体积比1:1混合的50mM NHS及200mM EDC活化，然后将抗人Fc片段抗体(购自Genway)用10mM的醋酸钠缓冲液(pH5.0)稀释至16.4μg/ml，与CM5芯片进行偶联反应。随后注入1M乙醇胺对剩余的活化位点进行封闭。待测抗体(即实施例2制备的全人源BLyS抗体)被芯片捕获后，注入7个不同浓度梯度稀释的实施例1制备的免疫原(即hBLyS-ECD)，通过Biacore仪器来检测抗体与抗原结合与解离。然后用Biacore X100Evaluation Software 2.0软件拟合得到解离常数与结合常数，亲和力常数为解离常数与结合常数的比值。结果如表14所示，说明BLyS抗体对hBLyS-ECD具有高亲和力。
表14 BLyS抗体对hBLyS-ECD的亲和常数
Figure PCTCN2017091839-appb-000016
实施例4检测BLyS纯化抗体阻断BLyS蛋白与其受体BAFF R的结合
通过BLyS蛋白的受体配体结合试验检测BLyS抗体阻断BLyS蛋白与其受体BAFF R的结合。
将BAFF R(购自R&D Systems)以50ng每孔加到96孔ELISA板中，塑料膜封好4℃孵育过夜。第二天用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次，加入封闭液[含有0.05％(v/v)Tween20和2％(w/w)BSA的PBS缓冲液]室温封闭1小时。倒掉封闭液，每孔先加入50μL实施例1和2所得的先导抗体，后加入实施例2制备的生物素化的hBLyS-ECD每孔50μL，混匀后37℃孵育1小时。用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。加入HRP(辣根过氧化物酶)标记的亲和素(购自Sigma)稀释液每孔100μL，37℃孵育1小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。每孔加入100μL TMB底物，室温孵育5分钟后，每孔加入100μL终止液(1.0N HCl)。用ELISA读板机(SpectraMax 384plus,Molecular Device)读取A450nm数值，结果如图9A～图9B和表15所示。表15说明，BLyS抗体能封闭BLyS蛋白与其受体BAFF R的结合。
表15-1 BLyS抗体阻断BLyS蛋白与其受体BAFF R的结合
表15-2 BLyS抗体阻断BLyS蛋白与其受体BAFF R的结合
Figure PCTCN2017091839-appb-000018
Figure PCTCN2017091839-appb-000019
实施例5小鼠B细胞增殖实验检测BLyS纯化抗体阻断人或鼠BLyS蛋白对B细胞增殖的作用
(一)小鼠脾脏分离B细胞
将新鲜获取的小鼠脾脏研磨后，用含有10％(w/w)胎牛血清的RPMI1640培养基(购自Invitrogen，货号：A10491)重悬，70μm细胞滤网(购自BD)过滤后，以1,500rpm转速4℃离心5分钟。弃上清，将细胞沉淀漩涡振荡15秒，加入红细胞裂解缓冲液(购自Sigma)，室温静置5分钟。用含有10％(w/w)胎牛血清的RPMI1640培养基补足到15mL，1500rpm转速4℃离心5分钟，得上清。上清用40μm细胞滤网(购自BD)过滤，得过滤后的上清，然后进行细胞计数。小鼠B细胞分离采用试剂盒(购自Miltenyi Biotec)，实验操作严格按照试剂盒说明书要求。具体实验简述如下：将过滤后的上清以300g转速4℃下离心10分钟，弃上清。每107个细胞中加入40μL缓冲液[含有0.5％(w/v)BSA和2mM EDTA的PBS缓冲液]和10μL该试剂盒中的结合非B细胞的生物素化的抗体混合物，混合后4℃孵育5分钟。每107个细胞中继续加入30μL缓冲液[含有0.5％(w/v)BSA和2mM EDTA的PBS缓冲液]和20μL抗生物素标记的磁珠(购自Miltenyi Biotec)，混合之后再4℃孵育10分钟，得细胞悬液。用缓冲液将体积补足到500μL。将LS分离柱(购自Miltenyi Biotec)放置在磁力架上，先加入3mL缓冲液润洗，再加入细胞悬液，收集流穿液，即未被标记的被富集的B细胞悬液。再加入3mL缓冲液，收集流穿液，并与上一步的流穿液合并，即得分离自小鼠脾脏的B细胞。
(二)小鼠B细胞增殖实验
将实施例5步骤(一)获得的B细胞以5╳104个细胞50μL每孔铺至96孔细胞培养板，加入2μg/ml的F(ab’)2片段化山羊抗小鼠IgM二抗(购自Jackson ImmunoResearch)、10ng/ml的实施例1制备的免疫原(hBLyS-ECD)或鼠BLyS蛋白(购自R&D Systems)和不同浓度的实施例1或2制备的先导抗体的溶液，保证每个反应孔100μL体积。将反应板于37℃、5％(v/v)CO2培养箱培养72小时后检测活细胞数。活细胞数目测定采用
Figure PCTCN2017091839-appb-000020
Luminescent Cell Viability Assay试剂盒(购自Promega)，实验操作严格按照试剂盒说明书要求。具体实验简述如下：在测定前将96孔板放在室温平衡30分钟，加入与细胞培养基等体积的
Figure PCTCN2017091839-appb-000021
试剂，放置在摇床2分钟以诱导细胞裂解，再将孔板放置在室温10分钟来稳定发光信号，最后用读板机(SpectraMax 384plus,Molecular  Device)读取数值。
检测BLyS抗体在实施例5步骤(二)所述实验中对小鼠B细胞增殖的影响。结果如图10～11和表16～17所示，图10说明，待测抗体可抑制hBLyS-ECD刺激引起的小鼠B细胞增殖。图11说明，待测抗体除L9G7可以抑制鼠BLyS诱导的小鼠B细胞增殖以外，与鼠BLyS无交叉反应。
表16 BLyS抗体对hBLyS-ECD刺激的小鼠B细胞增殖的抑制
Figure PCTCN2017091839-appb-000022
表17 BLyS抗体对鼠BLyS刺激的小鼠B细胞增殖的抑制
Figure PCTCN2017091839-appb-000023
实施例6 BLyS抗体结合抗原表位的鉴定
将纯化的全人源BLyS抗体和杂交瘤抗体进行竞争性酶联免疫吸附实验，分析不同抗体所结合的抗原表位。
先将纯化的BLyS抗体用PBS稀释到终浓度1.0μg/mL，然后以50μL每孔加到96孔ELISA板，用塑料膜封好4℃孵育过夜。第二天用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次，加入封闭液[含有0.05％(v/v)Tween20和1％(w/w)BSA的PBS缓冲液]，37℃封闭1小时。倒掉封闭液，将竞争性的抗体及IgG对照用PBS稀释到终浓度40μg/mL，然后以50μL每孔加到96孔ELISA板。再将生物素化的hBLyS-ECD用PBS稀释到终浓度2ng/mL，然后以50μL每孔加到96孔ELISA板。保证竞争性抗体以及hBLyS-ECD的终浓度分别为20μg/mL，1ng/mL。37℃孵育1小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。加入HRP(辣根过氧化物酶)标记的链霉亲和素(购自Sigma)，每孔100μL。37℃孵育1小时后，用洗板液[含有0.05％(v/v)Tween20的PBS缓冲液]洗板4次。每孔加入100μL TMB底物，室温孵育5分钟后，每孔加入100μL终止液(1.0N HCl)。用ELISA读板机(SpectraMax M5e，购自Molecular Device)读取A450nm数值。结果如表18A～表18B所示，说明BLyS抗体之间存在不同程度的竞争性，即结合不同的抗原表位。其中表中的数据为加入竞争性抗体后，对原抗体与hBLyS-ECD结合水平的抑制率(％)。
表18A BLyS全人源抗体竞争与hBLyS-ECD的结合
克隆号 2-1G11 L1D12 L9G7
2-1G11 82 91 79
L1D12 94 94 90
L9G7 96 97 95
表18B BLyS纯化抗体竞争与hBLyS-ECD的结合
Figure PCTCN2017091839-appb-000024
Figure PCTCN2017091839-appb-000025
实施例7轻重链可变区氨基酸序列测定及鼠-人嵌合抗体的制备
总RNA分离：将实施例1所选的先导抗体所对应的亚克隆培养所得的上清液检验过抗原结合后(即经过实施例3～5的检定和活性测定后)，通过离心搜集5×107个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟；加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中；加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清；加入1mL 75％(v/v)乙醇，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H2O溶解(55℃水浴促进溶剂10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于85℃反应10分钟终止反应。配置50μL PCR体系，包括1μL cDNA、每种引物25pmol、1μL DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，95℃预变性3分钟，95℃变性30秒，55℃退火30秒，72℃延伸35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒包括Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2017091839-appb-000026
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μL，缓冲液1μL，反应体系10μL，于16℃反应半小时得连接产物，其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μL连接产物加入100μL的感受态细胞(Ecos 101competent cells,购自Yeastern，货号FYE607)中，冰浴5分钟，而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μL无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μL涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养；次日，使用T载体上引物M13F和M13R配置30μL PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100μg/mL氨苄青霉素的LB固体培养皿上以保存菌株；PCR反应结束后，取出5μL进行 琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat，“Sequences of Proteins of Immunological Interest,”National Institutes of Health,Bethesda,Md.(1991)]。测序结果如表19～20所示。
本发明噬菌体制备的全人源BLyS抗体的克隆和测序请参见实施例2第(三)部分，表19、20也包含了这部分测序结果。
表19 BLyS抗体氨基酸序列编号
Figure PCTCN2017091839-appb-000027
其中，表19中的数字即为序列表中序列号，如2-1G11的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而2-1G11的重链蛋白可变区中CDR1的氨基酸序列为SEQ ID No.2。
表20 BLyS抗体基因核苷酸序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
2-1G11 105 106
L9G7 107 108
L1D12 109 110
35E6F7C3 111 112
8E7D9C7F5 113 114
20D1B6E9E5 115 116
78C11D2D12 117 118
89A2G5E7 119 120
97E7B3F2 121 122
97A3C2H4 123 124
67A2E1D10 125 126
111D10D6G3 127 128
93C6F10D3 129 130
其中，表20中的数字即为序列表中序列号，如编码2-1G11的重链蛋白可变区的氨基酸序列的核苷酸序列为SEQ ID No.105，而编码2-1G11的轻链蛋白可变区的氨基酸序列的核苷酸序列为SEQ ID No.106。
编码2-1G11的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.105中的第76位至第105位；
编码2-1G11的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.105中的第148位至第198位；
编码2-1G11的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.105中的第295位至第342位；
编码2-1G11的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.106中的第70位至第102位；
编码2-1G11的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.106中的第148位至第168位；
编码2-1G11的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.106中的第265位至第291位；
编码L9G7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.107中的第76位至第105位；
编码L9G7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.107中的第148位至第198位；
编码L9G7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.107中的第295位至第342位；
编码L9G7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.108中的第70位至第102位；
编码L9G7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.108中的 第148位至第168位；
编码L9G7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.108中的第265位至第291位；
编码L1D12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.109中的第76位至第105位；
编码L1D12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.109中的第148位至第195位；
编码L1D12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.109中的第292位至第330位；
编码L1D12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.110中的第67位至第99位；
编码L1D12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.110中的第145位至第165位；
编码L1D12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.110中的第262位至第297位。
编码35E6F7C3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.111中的第76位至第105位；
编码35E6F7C3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.111中的第148位至第198位；
编码35E6F7C3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.111中的第295位至第321位；
编码35E6F7C3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.112中的第70位至第99位；
编码35E6F7C3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.112中的第145位至第165位；
编码35E6F7C3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.112中的第262位至第288位。
编码8E7D9C7F5的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.113中的第76位至第105位；
编码8E7D9C7F5的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.113中的第148位至第198位；
编码8E7D9C7F5的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.113中的第295位至第342位；
编码8E7D9C7F5的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.114中的第70位至第114位；
编码8E7D9C7F5的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.114中的第160位至第180位；
编码8E7D9C7F5的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.114中的第277位至第303位。
编码20D1B6E9E5的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.115中的第76位至第105位；
编码20D1B6E9E5的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.115中的第148位至第198位；
编码20D1B6E9E5的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.115中的第295位至第339位；
编码20D1B6E9E5的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.116中的第70位至第117位；
编码20D1B6E9E5的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.116中的第163位至第183位；
编码20D1B6E9E5的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.116中的第280位至第306位。
编码78C11D2D12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.117中的第76位至第105位；
编码78C11D2D12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.117中的第148位至第198位；
编码78C11D2D12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.117中的第295位至第315位；
编码78C11D2D12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.118中的第70位至第99位；
编码78C11D2D12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.118中的第145位至第165位；
编码78C11D2D12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No. 118中的第262位至第288位。
编码89A2G5E7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.119中的第76位至第105位；
编码89A2G5E7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.119中的第148位至第195位；
编码89A2G5E7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.119中的第292位至第327位；
编码89A2G5E7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.120中的第70位至第105位；
编码89A2G5E7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.120中的第151位至第171位；
编码89A2G5E7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.120中的第268位至第294位。
编码97E7B3F2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.121中的第76位至第105位；
编码97E7B3F2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.121中的第148位至第198位；
编码97E7B3F2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.121中的第295位至第321位；
编码97E7B3F2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.122中的第70位至第117位；
编码97E7B3F2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.122中的第163位至第183位；
编码97E7B3F2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.122中的第280位至第306位。
编码97A3C2H4的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.123中的第76位至第105位；
编码97A3C2H4的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.123中的第148位至第198位；
编码97A3C2H4的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.123中的第295位至第327位；
编码97A3C2H4的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.124中的第70位至第102位；
编码97A3C2H4的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.124中的第148位至第168位；
编码97A3C2H4的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.124中的第265位至第291位。
编码67A2E1D10的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.125中的第76位至第105位；
编码67A2E1D10的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.125中的第148位至第198位；
编码67A2E1D10的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.125中的第295位至第333位；
编码67A2E1D10的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.126中的第70位至第102位；
编码67A2E1D10的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.126中的第148位至第168位；
编码67A2E1D10的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.126中的第265位至第291位。
编码111D10D6G3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.127中的第76位至第105位；
编码111D10D6G3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.127中的第148位至第198位；
编码111D10D6G3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.127中的第295位至第309位；
编码111D10D6G3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.128中的第70位至第102位；
编码111D10D6G3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.128中的第148位至第168位；
编码111D10D6G3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.128中的第265位至第291位；
编码93C6F10D3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.129 中的第76位至第105位；
编码93C6F10D3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.129中的第148位至第198位；
编码93C6F10D3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.129中的第295位至第336位；
编码93C6F10D3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.130中的第70位至第102位；
编码93C6F10D3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.130中的第148位至第168位；
编码93C6F10D3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.130中的第265位至第291位。
小鼠-人嵌合BLyS抗体的制备：根据上步的测序结果得到了抗体重链可变区和轻链可变区序列。小鼠-人嵌合BLyS抗体的生产和制备参考实施例2中的第(三)步骤，即；1、重组载体制备：将重链可变区定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤由上海睿智化学研究有限公司完成)，将轻链可变区定向克隆到包含信号肽和人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤由上海睿智化学研究有限公司完成)；2、细胞转染；3、抗体纯化。并对所获BLyS嵌合抗体进行特性鉴定(方法参见实施例3～5)。制得的各嵌合抗体命名在对应的先导抗体克隆号前加上首字符“c”，例如嵌合抗体c8E7D9C7F5对应的先导抗体克隆号为8E7D9C7F5。
结果如图12和表21所示，表21说明，嵌合抗体与BLyS重组蛋白在ELISA水平结合。其中表中的数据为OD450nm值。
表21 ELISA检测BLyS嵌合抗体与生物素化的hBLyS-ECD的结合反应
Figure PCTCN2017091839-appb-000028
通过Biacore仪器来检测抗体与抗原结合与解离。结果如表22所示，说明BLyS嵌 合抗体对hBLyS-ECD具有高亲和力。
表22 BLyS嵌合抗体对hBLyS-ECD的亲和常数
Figure PCTCN2017091839-appb-000029
结果如图13和表23所示。表23说明，BLyS嵌合抗体能封闭BLyS蛋白与其受体
BAFF R的结合。其中表中的数据为OD450nm值。
表23 BLyS嵌合抗体阻断BLyS蛋白与其受体BAFF R的结合
Figure PCTCN2017091839-appb-000030
结果如图14和表24所示。表24说明，BLyS嵌合抗体可以抑制hBLyS-ECD刺激引起的小鼠B细胞增殖。
表24 BLyS嵌合抗体对hBLyS-ECD刺激的小鼠B细胞增殖的抑制
Figure PCTCN2017091839-appb-000031
Figure PCTCN2017091839-appb-000032
实施例8小鼠体内检测BLyS抗体的中和活性
雌性BALB/c小鼠(8-9周龄，购自上海灵畅生物科技有限公司)接收后在SPF级饲养，适应1周后，开始实验。小鼠在第一天及第三天静脉注射实施例2和6制备的全人源或人鼠嵌合BLyS单克隆抗体(在免疫原注射1小时前)，克隆号分别为2-1G11、L1D12、c8E7D9C7F5、c20D1B6E9E5、c35E6F7C3、c97A3C2H4、c93C6F10D3和c111D10D6G3，剂量为3mg/kg。第一天到第四天每天通过皮下注射实施例1制备的免疫原(hBLyS-ECD)进行刺激诱导，剂量为0.3mg/kg。第五天将所有动物杀死，测量小鼠脾脏重量、B细胞在脾细胞中所占的比例和血清中IgA的的浓度。
部分实验结果见图15A～图15C和表25。IgG对照为人IgG。实验结果表明BLyS抗体能降低由hBLyS-ECD刺激所引起的小鼠脾细胞中B细胞比例的增加。
表25-1 BLyS抗体对hBLyS-ECD刺激后的小鼠B细胞在脾细胞中所占比例的影响
克隆号 B细胞在脾细胞中所占比例(％)
c8E7D9C7F5 34.80
c20D1B6E9E5 35.36
人IgG对照 51.05
表25-2 BLyS抗体对hBLyS-ECD刺激后的小鼠B细胞在脾细胞中所占比例的影响
克隆号 B细胞在脾细胞中所占比例(％)
2-1G11 25.64
c35E6F7C3 30.51
c97A3C2H4 24.10
人IgG对照 39.36
表25-3 BLyS抗体对hBLyS-ECD刺激后的小鼠B细胞在脾细胞中所占比例的影响
克隆号 B细胞在脾细胞中所占比例(％)
L1D12 26.75
c93C6F10D3 27.11
c111D10D6G3 33.24
c97A3C2H4 28.19
人IgG对照 38.29
实施例9人源化BLyS抗体的制备及鉴定
在Germline数据库中选取与上述嵌合抗体c8E7D9C7F5或c97A3C2H4的非CDR区匹配最好的人种系抗体重链和轻链可变区模板。人源化BLyS抗体的序列选自人种系外显子VH、JH、Vk和Jk序列。其中c8E7D9C7F5抗体重链可变区的模板为人种系抗体重链VH外显子的VH1-18，JH外显子的JH-6，轻链可变区的模板为人种系抗体轻链VK外显子的B3，JK外显子的JK-4。其中c97A3C2H4抗体重链可变区的模板为人种系抗体重链VH外显子的VH3-7，JH外显子的JH-6，轻链可变区的模板为人种系抗体轻链VK外显子的A10，JK外显子的JK-4。
根据Kabat定义确定的嵌合抗体c8E7D9C7F5或c97A3C2H4的重链和轻链CDR分别移植到所选人种系模板中，替换人种系模板的CDR区，得到人源化的抗体。然后，
以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VH和VL的构象有重要影响的构架区的残基进行回复突变，得到人源化之后的抗体。
人源化BLyS抗体变体的重链和轻链可变区与嵌合抗体的重链和轻链可变区的氨基酸序列比对如表26所示。其中人源化BLyS抗体h8E7D9C7F5变体的重链可变区序列分别为SEQ ID No.140，SEQ ID No.141，SEQ ID No.142，SEQ ID No.143，SEQ ID No.144，SEQ ID No.145，SEQ ID No.146，轻链可变区序列分别为SEQ ID No.147，SEQ ID No.148。人源化BLyS抗体h97A3C2H4变体的重链可变区序列分别为SEQ ID No.149，SEQ ID No.150，SEQ ID No.151，SEQ ID No.152，轻链可变区序列分别为SEQ ID No.153，SEQ ID No.154，SEQ ID No.155，SEQ ID No.156，SEQ ID No.157。
人种系重链可变区模板VH1-18/JH6(SEQ ID NO.136)
Figure PCTCN2017091839-appb-000033
人种系轻链可变区模板B3/JK4(SEQ ID NO.137)
Figure PCTCN2017091839-appb-000034
人种系重链可变区模板VH3-7/JH6(SEQ ID NO.138)
Figure PCTCN2017091839-appb-000035
人种系轻链可变区模板A10/JK4(SEQ ID NO.139)
Figure PCTCN2017091839-appb-000036
表26-1人源化BLyS抗体h8E7D9C7F5与嵌合抗体c8E7D9C7F5可变区的序列比对
Figure PCTCN2017091839-appb-000037
Figure PCTCN2017091839-appb-000038
Figure PCTCN2017091839-appb-000039
表26-2人源化BLyS抗体h97A3C2H4与嵌合抗体c97A3C2H4可变区的序列比对
Figure PCTCN2017091839-appb-000040
Figure PCTCN2017091839-appb-000041
人源化BLyS抗体变体的重链和轻链可变区的核苷酸序列如表27所示。其中人源化BLyS抗体h8E7D9C7F5变体的重链可变区核苷酸序列分别为SEQ ID No.158，SEQ ID No.159，SEQ ID No.160，SEQ ID No.161，SEQ ID No.162，SEQ ID No.163，SEQ ID No.164，轻链可变区核苷酸序列分别为SEQ ID No.165，SEQ ID No.166。人源化BLyS抗体h97A3C2H4变体的重链可变区核苷酸序列分别为SEQ ID No.167，SEQ ID No.168，SEQ ID No.169，SEQ ID No.170，轻链可变区核苷酸序列分别为SEQ ID No.171，SEQ ID No.172，SEQ ID No.173，SEQ ID No.174，SEQ ID No.175。
表27-1人源化BLyS抗体h8E7D9C7F5可变区核苷酸的序列编号
Figure PCTCN2017091839-appb-000042
Figure PCTCN2017091839-appb-000043
表27-2人源化BLyS抗体h97A3C2H4可变区核苷酸的序列编号
抗体名称 重链可变区 轻链可变区
h97A3C2H4-1 167 171
h97A3C2H4-2 167 172
h97A3C2H4-3 168 171
h97A3C2H4-4 168 172
h97A3C2H4-5 169 171
h97A3C2H4-6 169 172
h97A3C2H4-7 169 173
h97A3C2H4-8 169 174
h97A3C2H4-9 169 175
h97A3C2H4-10 170 173
h97A3C2H4-11 170 174
h97A3C2H4-12 170 175
根据人源化VH或VL结构域合成的重叠寡核苷酸，利用PCR重叠延伸来组装各结构域。利用掺入PCR产物的限制性位点将VH结构域定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤由上海睿智化学研究有限公司完成)，将VL结构域定向克隆到包含信号肽和人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤由上海睿智化学研究有限公司完成)中，得到的重组质粒经过测序验证，抽提高纯度的重组质粒，经0.22μm滤膜过滤，供转染使用。人源化BLyS抗体的生产和制备参考实施例2中的第(三)步骤，并对所获BLyS抗体进行特性鉴定(见实施例3～5，7)。
结果如图16和表28所示，表28说明，人源化抗体与BLyS重组蛋白在ELISA水平结合。其中表中的数据为OD450nm值。
表28 ELISA检测人源化抗体与生物素化的hBLyS-ECD的结合反应
Figure PCTCN2017091839-appb-000044
通过Biacore仪器来检测抗体与抗原结合与解离。结果如表29所示，说明BLyS人源化抗体对hBLyS-ECD具有高亲和力。
表29 BLyS人源化抗体对hBLyS-ECD的亲和常数
Figure PCTCN2017091839-appb-000045
结果如图17和表30所示。表30说明，BLyS人源化抗体能封闭BLyS蛋白与其受体BAFF R的结合。其中表中的数据为OD450nm值。
表30 BLyS人源化抗体阻断BLyS蛋白与其受体BAFF R的结合
Figure PCTCN2017091839-appb-000046
结果如图18和表31所示。表31说明，BLyS人源化抗体可以抑制hBLyS-ECD刺激引起的小鼠B细胞增殖。
表31 BLyS人源化抗体对hBLyS-ECD刺激的小鼠B细胞增殖的抑制
Figure PCTCN2017091839-appb-000047
同时，在小鼠体内检测BLyS人源化抗体的中和活性。实验结果见图19A～19B和表32。实验结果表明BLyS人源化抗体能降低由hBLyS-ECD刺激所引起的小鼠脾细胞中B细胞比例的增加，同时有效抑制血清中IgA水平。
表32-1 BLyS人源化抗体对hBLyS-ECD刺激后的小鼠B细胞在脾细胞中所占比例的影响
克隆号 B细胞在脾细胞中所占比例(％)
h8E7D9C7F5-1 28.29
h97A3C2H4-9 28.98
人IgG对照 39.74
表32-2 BLyS人源化抗体对小鼠血清中IgA浓度的影响
克隆号 IgA浓度(μg/mL)
h8E7D9C7F5-1 80.89
h97A3C2H4-9 121.33
人IgG对照 387.02
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (20)

  1. 一种分离的蛋白质，其特征在于，其包括BLyS抗体的互补决定区(CDR)：重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，BLyS抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82、SEQ ID No.90或SEQ ID No.98所示；所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83、SEQ ID No.91或SEQ ID No.99所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84、SEQ ID No.92或SEQ ID No.100所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86、SEQ ID No.94或SEQ ID No.102所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87、SEQ ID No.95或SEQ ID No.103所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88、SEQ ID No.96或SEQ ID No.104所示；
    或者，所述重链CDR1的氨基酸序列如与序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82、SEQ ID No.90或SEQ ID No.98所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列如与序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83、SEQ ID No.91或SEQ ID No.99所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列如与序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID  No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84、SEQ ID No.92或SEQ ID No.100所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR1的氨基酸序列如与序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86、SEQ ID No.94或SEQ ID No.102所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列如与序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87、SEQ ID No.95或SEQ ID No.103所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列如与序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88、SEQ ID No.96或SEQ ID No.104所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
  2. 如权利要求1所述的蛋白质，其特征在于，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.66所示，所述重 链CDR2的氨基酸序列如序列表SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.68所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.76所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.82所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.83所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.84所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.90所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.91所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.92所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.98所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.99所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.100所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.72所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.80所示；所述轻链CDR1 的氨基酸序列如序列表SEQ ID No.86所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.87所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.88所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.94所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.95所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.96所示；或者，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.102所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.103所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.104所示。
  3. 如权利要求1或2所述的蛋白质，其特征在于，其还包括BLyS抗体的构架区，所述构架区包括重链构架区和/或轻链构架区；较佳地，所述重链构架区为人或鼠抗体重链构架区，和/或，所述轻链构架区为人或鼠抗体轻链构架区；更佳地，所述重链构架区为人抗体重链构架区，优选为人种系抗体重链VH外显子的VH1-18、VH外显子的VH3-7或者JH外显子的JH-6，且所述轻链构架区为人抗体轻链构架区，优选为人种系抗体轻链VK外显子的B3、JK外显子的JK-4或者VK外显子的A10。
  4. 如权利要求3所述的蛋白质，其特征在于，其包括所述CDR和构架区组成的BLyS抗体的重链可变区和/或BLyS抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65、SEQ ID No.73、SEQ ID No.81、SEQ ID No.89、SEQ ID No.97、SEQ ID NO.140、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151或SEQ ID NO.152所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85、SEQ ID No.93、SEQ ID No.101、SEQ ID No.147、SEQ ID No.148、SEQ ID No.153、SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、或SEQ ID No.157所示。
  5. 如权利要求4所述的蛋白质，其特征在于，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示，且所述轻链 可变区的氨基酸序列如序列表SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.65所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.69所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.77所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.81所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.85所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.89所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.93所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.97所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.101所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.140所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.140所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.144所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.144所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.145所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.145所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.146所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.147所示；所述重链可变区的氨基酸序列如序 列表SEQ ID No.146所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.149所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.149所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.154所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.150所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.150所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.154所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.151所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.151所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.154所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.151所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.155所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.151所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.156所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.151示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.157所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.152所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.155所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.152所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.156所示；或者所述重链可变区的氨基酸序列如序列表SEQ ID No.152所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.157所示。
  6. 如权利要求1～5中任一项所述的蛋白质，其特征在于，所述的蛋白质还包括抗体重链恒定区和/或抗体轻链恒定区。
  7. 如权利要求6所述的蛋白质，其特征在于，所述的抗体重链恒定区为人源或小鼠源抗体重链恒定区；所述的抗体轻链恒定区为人源或小鼠源抗体轻链恒定区。
  8. 如权利要求7所述的蛋白质，其特征在于，所述的抗体重链恒定区为人源抗体重链恒定区；所述的抗体轻链恒定区为人源抗体轻链恒定区。
  9. 如权利要求1～5任一项所述的蛋白质，其特征在于，所述的蛋白质是BlyS抗体的单克隆抗体、抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体或单区抗体。
  10. 一种核酸，其特征在于，其编码如权利要求1～9中任一项所述的蛋白质。
  11. 如权利要求10所述的核酸，其特征在于，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.105、SEQ ID No.107、SEQ ID No.109、SEQ ID No.111、SEQ ID  No.113、SEQ ID No.115、SEQ ID No.117、SEQ ID No.119、SEQ ID No.121、SEQ ID No.123、SEQ ID No.125、SEQ ID No.127、SEQ ID No.129、SEQ ID NO.158、SEQ ID NO.159、SEQ ID NO.160、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.163、SEQ ID NO.164、SEQ ID NO.167、SEQ ID NO.168、SEQ ID NO.169或SEQ ID NO.170所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106、SEQ ID No.108、SEQ ID No.110、SEQ ID No.112、SEQ ID No.114、SEQ ID No.116、SEQ ID No.118、SEQ ID No.120、SEQ ID No.122、SEQ ID No.124、SEQ ID No.126、SEQ ID No.128、SEQ ID No.130、SEQ ID No.165、SEQ ID No.166、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173、SEQ ID No.174或SEQ ID No.175所示。
  12. 如权利要求11所述的核酸，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.105所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.106所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.107所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.108所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.109所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.110所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.112所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.114所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.116所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.118所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.119所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.120所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.121所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.122所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.123所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.124所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.125所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.126所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.127所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.128所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.129所示，且编码所述轻 链可变区的核酸的核苷酸序列如序列表SEQ ID No.130所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.158所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.158所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.159所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.159所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.160所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.160所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.161所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.161所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.165所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.166所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.167所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.167所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.168所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.168所示，且编码所述轻链可变区的 核酸的核苷酸序列如序列表SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.169所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.169所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.169所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.169所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.169所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.175所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.170所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.170所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.174所示；或者，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.170所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.175所示。
  13. 一种包含如权利要求10～12中任一项所述的核酸的重组表达载体。
  14. 一种包含如权利要求13所述的重组表达载体的重组表达转化体。
  15. 一种BlyS抗体的制备方法，其包括如下步骤：培养如权利要求14所述的重组表达转化体，从培养物中获得BlyS抗体。
  16. 一种检测过表达BlyS蛋白的细胞的方法，其特征在于，包括如下的步骤：如权利要求1～9中任一项所述的蛋白质与待检样品在体外接触，检测如权利要求1～9中任一项所述的蛋白质与所述待检样品的结合即可。
  17. 一种检测过表达BlyS蛋白的细胞的组合物，其特征在于，其包括如权利要求1～9中任一项所述的蛋白质作为活性成分。
  18. 一种药物组合物，其特征在于，其包括如权利要求1～9中任一项所述的蛋白质作为活性成分和药学可接受的载体。
  19. 如权利要求18所述的药物组合物，其特征在于，所述的药物组合物包括0.01～99.99％的如权利要求1～9中任一项所述的蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
  20. 一种如权利要求1～9中任一项所述的蛋白质或者如权利要求18或19所述的药物组合物在制备预防或治疗与BLyS表达或功能异常相关的疾病的药物中的应用；较佳 地，所述的与BLyS表达或功能异常相关的疾病为自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病。
PCT/CN2017/091839 2016-07-06 2017-07-05 一种BLyS抗体及其制备方法和应用 WO2018006824A1 (zh)

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WO2022223028A1 (zh) * 2021-04-23 2022-10-27 上海君实生物医药科技股份有限公司 抗BLyS抗体、其药物组合物及其用途

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