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WO2019109947A1 - 抗人il6单克隆抗体及其制备方法和用途 - Google Patents

抗人il6单克隆抗体及其制备方法和用途 Download PDF

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Publication number
WO2019109947A1
WO2019109947A1 PCT/CN2018/119353 CN2018119353W WO2019109947A1 WO 2019109947 A1 WO2019109947 A1 WO 2019109947A1 CN 2018119353 W CN2018119353 W CN 2018119353W WO 2019109947 A1 WO2019109947 A1 WO 2019109947A1
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amino acid
acid sequence
variable region
chain variable
seq
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PCT/CN2018/119353
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English (en)
French (fr)
Inventor
殷刘松
林峰
王磊
许云云
丁力
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南京金斯瑞生物科技有限公司
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Priority to EP18886699.0A priority Critical patent/EP3722311A4/en
Priority to US16/770,308 priority patent/US20220396615A1/en
Publication of WO2019109947A1 publication Critical patent/WO2019109947A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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 tumor immunotherapy and molecular immunology, in particular to monoclonal antibodies against human IL6.
  • the invention also relates to a process for the preparation of the anti-human IL6 monoclonal antibody and to the use thereof.
  • the immune system is a host defense system. In order to function normally, the immune system must be able to sensitively detect the invasion of foreign pathogens and distinguish them from the healthy tissues of the organism itself.
  • the vertebrate immune system is a functional system composed of various organs, tissues, cells and molecules. It is the most effective mechanism for the body to defend against the invasion of foreign substances. These immune organs, tissues, cells and molecules cooperate and balance each other. In order to protect the body from external infection and maintain homeostasis (Janeway et al., Immunology: The Immune System in Health and Disease. New York: Garland Science, 2005). This mutual cooperation and mutual checks and balances require the coordination of numerous immunological checkpoint proteins and cytokines.
  • the stimulating immune checkpoint protein enhances the immune system's defense response, and the inhibitory immune checkpoint protein controls the immune system to prevent autoimmune reactions.
  • Carcinogenesis is the result of normal somatic cells losing normal cell regulatory functions and gene mutations accumulating to a certain extent.
  • tumor immunotherapy is widely used, and it has become one of the most important means of cancer treatment. Tumor immunotherapy can enhance the body's immune function, so that human T cells can better recognize cancerous cells, thereby killing cancer cells.
  • the antibody Pembrolizumab against the inhibitory immunological checkpoint protein PD1 and the Ipilimumab antibody against CTLA4 have been approved for the treatment of various cancer indications.
  • the clinical benefits of these immunological checkpoint-based antibody drugs are still very limited.
  • Interleukins the most important class of cytokines, have a variety of immunomodulatory functions that direct the maturation, differentiation, migration and adhesion of immune system cells. In tumorigenesis, these cytokines directly stimulate immune effector and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells (Yoshimoto et al., Immunotherapy 5: 825-844, 2009). Recent studies have shown that interleukins are involved in many tumor-associated molecular mechanisms and have been utilized to develop many cytokine-based cancer therapies.
  • tumor cells have a variety of methods for immune escape, including the creation of a safe tumor microenvironment, secretion of cytokines and anti-inflammatory cytokines that suppress the immune system, and the recruitment or transformation of inflammatory cells that suppress immune responses, including regulatory T. (Treg) cells, bone marrow-derived suppressor cells (MDSC) and dendritic cell (DC) (Stewart et al., Cancer Metastasis Rev. 30: 125-140, 2011).
  • T. regulatory T.
  • MDSC bone marrow-derived suppressor cells
  • DC dendritic cell
  • Members of the interleukin family exist in the tumor microenvironment to interact with various biomolecules such as cancer stem cells, microRNAs, and epithelial mesenchymal cells. Therefore, efficient cancer immunotherapy can be developed based on the principle that interleukin affects the mechanism of tumor formation.
  • interleukin 2 IL2
  • interleukin 6 IL6
  • interleukin 7 IL7
  • interleukin 12 IL12
  • interleukin 18 IL18
  • Related therapies for interleukin 21 IL21 have entered the clinical trial phase of patients.
  • Interleukin-6 has multiple functions in the body, which can cause regulatory T cell (Treg) imbalance and secretion of various inflammatory substances.
  • Treg regulatory T cell
  • IL6 is capable of producing CD44-positive cells with stem cell traits by inducing epithelial stromal cell switching in breast cancer cells.
  • the level of IL6 in tumor-associated endothelial cells is directly related to the tumorigenicity of cancer stem cells (Int. J. Mol. Sci. 16: 1691-1710, 2015).
  • IL6 signaling is a potential therapeutic strategy for cancer.
  • Johnson & Johnson's anti-human IL6 Siltuximab antibody was approved by the US Food and Drug Administration for the treatment of Castleman's disease in 2014 and is undergoing a clinical phase II trial of multiple myeloma.
  • no antibodies against human IL6 have been marketed for tumor immunotherapy.
  • different anti-human IL6 monoclonal antibodies also have different degrees of side reactions, including induction of immunogenicity in certain patients, and different degrees of developability of different IL6 monoclonal antibodies. Therefore, there is a need to develop new functional antibodies that block IL6 signaling, which should have higher affinity, specificity, functionality, and diversity.
  • the invention provides an anti-human IL-6 monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, said light chain
  • the variable region includes LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from one of the following combinations:
  • amino acid sequence of HCDR2 is YINPITGYTENNQKFKD;
  • HCDR3 The amino acid sequence of HCDR3 is GIRGFTY;
  • the amino acid sequence of LCDR1 is RASENVDNSDNSFMH;
  • amino acid sequence of LCDR2 is RASNLDS
  • amino acid sequence of LCDR3 is QQTNEAPLT
  • amino acid sequence of HCDR2 is YVIPSTGYTDYNQSFKD;
  • the amino acid sequence of HCDR3 is LLPGFAY;
  • the amino acid sequence of LCDR1 is RSSQSLVDSNGNTYLH;
  • amino acid sequence of LCDR2 is KVSNRFS
  • amino acid sequence of LCDR3 is SQSTHVPPT
  • amino acid sequence of HCDR2 is YIDPRTASIYYNQKFKD;
  • amino acid sequence of HCDR3 is ILYGKYDV
  • the amino acid sequence of LCDR1 is RSSQSLVDSNGNTYLH;
  • amino acid sequence of LCDR2 is KVSNRFS
  • amino acid sequence of LCDR3 is SQSTHVPPT
  • HCDR2 The amino acid sequence of HCDR2 is EIRSKTYHPATYYTKSVRG;
  • amino acid sequence of HCDR3 is PRYYGGYFDY;
  • the amino acid sequence of LCDR1 is RASESVDNYGMSFMN;
  • the amino acid sequence of LCDR2 is TASNQGS
  • amino acid sequence of LCDR3 is QQSKEVPYT
  • HCDR2 The amino acid sequence of HCDR2 is AHIPGNGDTSYSQKFKD;
  • amino acid sequence of HCDR3 is GDAGYSAWFAY;
  • amino acid sequence of LCDR1 is SASESVDSYGNNFMH;
  • the amino acid sequence of LCDR2 is LASKLES;
  • the amino acid sequence of LCDR3 is QQNNEDPLT;
  • amino acid sequence of HCDR2 is KMWSNGDTDYDSAIRS
  • amino acid sequence of HCDR3 is YYFSSYGGGYFDY;
  • amino acid sequence of LCDR1 is RASKSVSTYMH;
  • amino acid sequence of LCDR2 is SASNLES
  • amino acid sequence of LCDR3 is QQSDELPDT
  • amino acid sequence of HCDR2 is TISPSGGTSYSRDSVKG;
  • amino acid sequence of HCDR3 is ERIYNTYFDY;
  • the amino acid sequence of LCDR1 is LPSEDISSDLA
  • the amino acid sequence of LCDR2 is NANTLPN;
  • the amino acid sequence of LCDR3 is QQYDSYPYT.
  • amino acid sequence of the heavy chain variable region and the light chain variable region of the anti-human IL-6 monoclonal antibody is selected from one of the following combinations:
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 5
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27.
  • the dissociation constant KD between the anti-human IL6 monoclonal antibody and IL6 is less than 10 nM. In a more preferred embodiment, the dissociation constant KD between the anti-human IL6 monoclonal antibody and IL6 is less than 1 nM.
  • the anti-human IL6 monoclonal antibody inhibits the proliferation promoting effect of IL6 on TF-1 cells.
  • the invention provides an isolated polynucleotide encoding the anti-human IL6 monoclonal antibody.
  • the polynucleotide comprises a heavy chain variable region coding sequence encoding a heavy chain variable region of the anti-human IL6 monoclonal antibody and a light chain encoding the anti-human IL6 monoclonal antibody
  • the light chain variable region coding sequence of the variable region, the heavy chain variable region coding sequence and the light chain variable region coding sequence being selected from one of the following combinations:
  • the heavy chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2, and the light chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 4;
  • the heavy chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 6, and the light chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 8;
  • the heavy chain variable region coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 10, and the light chain variable region coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 12;
  • the heavy chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 14, and the light chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 16;
  • the heavy chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 18, and the light chain variable region coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 20;
  • the heavy chain variable region coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 22, and the light chain variable region coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 24;
  • the heavy chain variable region coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 26, and the light chain variable region coding sequence includes the nucleotide sequence set forth in SEQ ID NO: 28.
  • the invention provides an expression vector comprising the polynucleotide described above.
  • the invention provides a host cell comprising the expression vector described above.
  • the host cell is a HEK293-6E cell.
  • the invention provides a method of making an anti-human IL6 monoclonal antibody comprising transfecting competent cells with the expression vector described above and culturing the cells.
  • the invention provides the use of the anti-human IL6 monoclonal antibody, the polynucleotide, the expression vector, the host cell in the preparation of a medicament for anti-tumor.
  • the tumor is selected from the group consisting of multiple myeloma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma, prostate cancer, breast cancer, and ovarian cancer.
  • the invention provides an anti-tumor pharmaceutical composition
  • an anti-tumor pharmaceutical composition comprising an effective amount of the anti-human IL6 monoclonal antibody and a pharmaceutically acceptable carrier.
  • the invention provides a method of making an anti-human IL6 monoclonal antibody, comprising:
  • the spleen cells of the mouse are fused with myeloma cells, and the obtained hybridoma cells are screened to obtain a positive mother clone which specifically recognizes human IL6;
  • the anti-IL6 monoclonal antibody provided by the invention has high affinity, high specificity to IL6, and can recognize different epitopes of IL6, respectively, can stimulate or inhibit the downstream pathway of IL6, activate or inhibit T cell secretion of cytokines, for example, Inhibition of IL6 downstream pathways inhibits TF-1 cell proliferation. Therefore, the functional monoclonal antibody against IL6 provided by the present invention can inhibit cell proliferation by blocking the IL6 signaling pathway, thereby achieving the purpose of tumor immunotherapy.
  • Figure 1 shows the results of serum ELISA titer detection in mice immunized with human IL6.
  • Figure 2 shows the results of serum ELISA titer assays in rats immunized with human IL6.
  • Figure 3 is a graph showing the specific binding of the anti-IL6 monoclonal antibody of the present invention to the IL6 recombinant protein.
  • Figure 4 is a graph showing that the anti-IL6 monoclonal antibody of the present invention specifically inhibits the proliferation of TF-1 cells.
  • Figure 5 shows the results of measuring the affinity of the anti-IL6 monoclonal antibody of the present invention for IL6 protein.
  • antibody refers to an immunoglobulin molecule, which is typically a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds.
  • the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus depending on the conservation difference of the amino acid sequence.
  • V variable region
  • C constant region
  • three local regions each have a higher degree of amino acid composition and alignment, which is a key position for the binding of the antibody to the antigen, and is therefore also referred to as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • the three heavy chain complementarity determining regions are referred to as HCDR1, HCDR2 and HCDR3, respectively, and the three light chain complementarity determining regions are referred to as LCDR1, LCDR2 and LCDR3, respectively.
  • the variable regions of one heavy chain and one light chain interact to form an antigen binding site (Fv).
  • Antibodies can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses, for example, IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art.
  • the invention is intended to include antibodies of any of the foregoing classes or subclasses.
  • antibody as used herein is also intended to encompass a digested fragment or a functional variant thereof, for example, an antibody fragment capable of binding to IL6 or a portion thereof, including but not limited to a Fab (eg, obtained by papain digestion of an antibody), F (ab') 2 (for example, obtained by pepsin digestion), Fv or scFv (for example, obtained by molecular biology techniques).
  • Fab eg, obtained by papain digestion of an antibody
  • F (ab') 2 for example, obtained by pepsin digestion
  • Fv or scFv for example, obtained by molecular biology techniques.
  • the term "monoclonal antibody” as used herein refers to a homogeneous antibody that is directed only to a particular antigenic epitope. Each monoclonal antibody is directed against a single antigenic determinant on the antigen as compared to a conventional polyclonal antibody preparation that typically includes different antibodies directed against different antigenic determinants (epitopes).
  • the modifier “monoclonal” refers to the uniform character of an antibody and is not to be construed as requiring an antibody produced by any particular method.
  • the monoclonal antibodies of the invention are preferably produced by recombinant DNA methods or by screening methods described elsewhere herein.
  • isolated polynucleotide refers to a polynucleotide that is naturally occurring in nature, including polynucleotides isolated from nature (including organisms) by biological techniques, as well as synthetic polynucleotides. .
  • the isolated polynucleotide can be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof.
  • Provided herein are a plurality of nucleotide sequences for encoding the heavy chain variable region and the light chain variable region of an anti-human IL6 monoclonal antibody, it being noted that one skilled in the art can according to the heavy chain provided herein.
  • amino acid sequences of the variable region and the light chain variable region are designed to have nucleotide sequences that are not identical to the nucleotide sequences provided above, but all encode the same amino acid sequence. These altered nucleotide sequences are also included within the scope of the invention.
  • vector refers to any molecule (eg, nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide-encoding information into a host cell.
  • expression vector or “expression cassette” refers to a vector suitable for expressing a gene of interest (a nucleotide sequence to be expressed) in a host cell, and typically includes a gene of interest, a promoter, a terminator, a marker gene, and the like.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a gene of interest of interest.
  • the term includes progeny of the parental cell, whether or not the progeny is identical in morphology or genetic composition to the original parental cell, as long as the progeny are present in the selected gene of interest.
  • Common host cells include bacteria, yeast, mammalian cells, and the like.
  • transfection refers to the ingestion of foreign or exogenous DNA by a cell which can be used to introduce one or more exogenous DNA portions into a suitable host cell.
  • the cells can be induced by physicochemical methods (e.g., by treatment with calcium chloride) to be in a physiological state that is optimal for uptake and to accommodate foreign DNA, i.e., "competent.”
  • ⁇ ективное amount refers to an amount that produces a function or activity to a human and/or animal and that is acceptable to humans and/or animals.
  • “Pharmaceutically acceptable carrier” means a carrier for administration, including various excipients, diluents, buffers, and the like, which are suitable for administration to humans and/or animals without excessive adverse side reactions while It is suitable for maintaining the viability of a drug or active agent located therein.
  • Example 1 Obtainment of human IL6 hybridoma cell line
  • the antigen was tag-free human IL6 recombinant protein (GenScript, Cat. No. Z03034).
  • Female Balb/c and C57bl/6 mice and Wistar rats were immunized subcutaneously with a 1:1 emulsion of 50 ⁇ g of IL6 protein in 200 ⁇ l of Freund's complete adjuvant (Sigma-Aldrich). Subsequently, mice were boosted by intraperitoneal/subcutaneous injection of a 1:1 emulsion of Freund's incomplete adjuvant (Sigma-Aldrich) containing 25 ⁇ g of IL6 for up to 3 times every two weeks.
  • the spleen was extracted and homogenized to produce a single cell suspension, while a single cell suspension of myeloma cells (SP2/0) was prepared. 8.9 ⁇ 10 7 splenocytes were fused with 4.1 ⁇ 10 7 SP2/0 mouse myeloma cells using electrofusion. The fused cells were resuspended in 100 ml of DMEM/10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine and aminoguanidine, and pipetted into a 96-well plate in a volume of 100 ⁇ l. . The plates were incubated at 37 ° C in 6% CO 2 . After 7 days of incubation, the presence of antibodies against IL6 was tested using the ELISA binding described below.
  • ELISA binding assay Indirect ELISA was used to assess the binding capacity of antibodies in the supernatant to IL6.
  • ELISA plates (Nunc) were coated with 0.5 ⁇ g/ml of recombinant IL6 protein in 100 ⁇ l/well of PBS overnight at 4 °C. The plates were washed with PBS-T (0.05% Tween) and blocked with 200 ⁇ l/well of PBST containing 1% BSA for 0.5 hour at 37 °C. Subsequently, the blocking solution was discarded, and 100 ⁇ l of the hybridoma cell culture supernatant was added to each plate, followed by incubation at room temperature for 1 hour.
  • the plates were washed three times with PBST and incubated with 100 ⁇ l/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fab-specific) (GenScript) for 0.5 hours at 37 °C.
  • the plates were washed five times with PBST, then TMB chromogenic solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark.
  • the reaction was stopped by the addition of 50 ⁇ l of 1 M HCl stop solution (Sigma).
  • the plate was read at 450 nm using a microplate reader.
  • Subcloning was performed using the limiting dilution method.
  • the cells were serially diluted using a hemocytometer and in a DMEM/10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine and aminoguanidine until the cell density reached 5-15. Cells/ml.
  • 200 ⁇ l of the cell solution was pipetted into 96 wells at a density of 1-3 cells/well. After the culture was incubated at 37 ° C for 1 week in 5% CO 2 , the supernatant was subjected to the above ELISA binding test to evaluate the presence of antibodies against IL6.
  • TRIzol (Ambion) was used from 3 ⁇ 10 6 - 5 ⁇ 10 6 extraction of total hybridoma RNA, and using an antibody subtype-specific primers and universal primers (PrimeScript TM 1stStrand cDNA Synthesis Kit, Takara) which is reverse transcribed into cDNA.
  • the murine immunoglobulin heavy and light chain V-region fragments were subsequently amplified by RACE PCR (GenScript), and the resulting PCR fragment was subcloned into the pMD18-T vector system (Takara), and the vector-specific primer pair insert was used. Sequencing was performed. The unique V-region nucleotide/amino acid sequences of clones 8H10B7E6, 23A9H3, 29D6B5, 41A10B8, 49F10H6, 53A2F9 (rat), 61G1D8 (rat) were finally obtained. In the amino acid sequences of the heavy and light chains, the CDR1 region is underlined, the CDR2 region is underlined, and the CDR3 region is wavy underlined.
  • 8H10B7E6 heavy chain variable region nucleotide sequence SEQ ID NO: 2
  • 29D6B5 heavy chain variable region amino acid sequence SEQ ID NO:9
  • 29D6B5 heavy chain variable region nucleotide sequence SEQ ID NO: 10
  • 29D6B5 light chain variable region amino acid sequence: SEQ ID NO: 11
  • 29D6B5 light chain variable region nucleotide sequence: SEQ ID NO: 12
  • 61G1D8 heavy chain variable region amino acid sequence SEQ ID NO: 25
  • 61G1D8 heavy chain variable region nucleotide sequence SEQ ID NO:26
  • 61G1D8 light chain variable region nucleotide sequence SEQ ID NO: 28
  • a DNA fragment comprising a light chain variable region + constant region and a heavy chain variable region + constant region was separately synthesized and inserted into a pTT5 expression vector to form an expression plasmid.
  • the above expression plasmid was co-transfected into HEK293-6E cells and cultured in a shake flask at 37 ° C for 10 days, and the supernatant was collected for antibody purification. Prior to purification, the tubing and Protein A column were depyrogenated with 0.2 M NaOH. The column was re-equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2 x of the above buffer and sterilized by filtration.
  • the filtered supernatant and Protein A column were incubated for 2 hours at room temperature, and after washing the column with 1 ⁇ of the above buffer, the IgG was eluted with sterile 0.1 M sodium citrate (pH 3.5), and the eluate was collected and used. One part by volume of sterile 1 M Tris-HCl (pH 9) was neutralized. The product buffer was exchanged to PBS (pH 7.4) under sterile conditions to remove any elution buffer and concentrate the sample. After concentration, the antibody was quantified by OD280 nm using an extinction coefficient Ec (0.1%) of 1.43.
  • Purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system using 10% pre-formed gel (GenScript). The gel was stained with Esta 2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (GenScript).
  • ELISA plates (Nunc) were coated with 0.5 ⁇ g/ml of recombinant IL6 protein in 100 ⁇ l/well of PBS overnight at 4 °C. The plates were washed with PBS-T (0.05% Tween) and blocked with 200 ⁇ l/well of PBST containing 1% BSA for 0.5 hour at 37 °C. Subsequently, the blocking solution was discarded, 100 ⁇ l of 10 ⁇ g/ml of purified antibody was added to the well, and diluted by a 3-fold gradient for a total of 11 test concentration gradients. It was then incubated for 1 hour at room temperature.
  • the plates were washed three times with PBST and incubated with 100 ⁇ l/well of horseradish peroxidase-conjugated goat anti-mouse/rat IgG (Fab-specific) (GenScript) for 0.5 hours at 37 °C.
  • the plates were washed five times with PBST, then TMB chromogenic solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark.
  • the reaction was stopped by the addition of 50 ⁇ l of 1 M HCl stop solution (Sigma).
  • the plate was read at 450 nm using a microplate reader.
  • the binding ability of clones 8H10B7E6, 23A9H3, 29D6B5, 41A10B8, 49F10H6, 53A2F9 (rat), 61G1D8 (rat) for recombinant protein IL6 is shown in FIG.
  • a competition ELISA was used to assess the epitope of the purified antibody.
  • ELISA plates (Nunc) were coated overnight at 4 °C with 100 ⁇ l/well of 0.5 ⁇ g/ml recombinant IL6 in PBS. The plates were washed with PBS-T (0.05% Tween) and blocked with 200 ⁇ l/well of PBST containing 1% BSA for 0.5 hour at 37 °C. Subsequently, the blocking solution was discarded, and a pair of one (one of the labeled biotin) was added to each well for the test antibody to be tested for competition, 100 ⁇ l (10 ⁇ g/ml) of each purified antibody. It was then incubated for 1 hour at 37 °C.
  • the plates were washed 3 times with PBST and incubated with 100 ⁇ l/well of streptavidin HRP (SA-HRP, GenScript) for 10 minutes at 37 °C.
  • the plates were washed five times with PBST, then TMB chromogenic solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark.
  • the reaction was stopped by the addition of 50 ⁇ l of 1 M HCl stop solution (Sigma).
  • the plate was read at 450 nm using a microplate reader.
  • TF-1 cells The growth of TF-1 cells is dependent on granulocyte macrophage colony-stimulating factor (GM-CSF). If the factor is lacking or its function is blocked, cell proliferation, differentiation and maturation will be significantly inhibited.
  • the monoclonal antibody function test is to replace the GM-CSF in the culture medium with the IL6 protein having the same function, and then determine the number of TF-1 living cells after a certain period of time by adding the anti-IL6 monoclonal antibody. Whether monoclonal antibodies block the biological function of IL-6 protein.
  • TF-1 cells were cultured normally in medium containing GM-CSF, and the cells were collected before the experiment, washed twice with basal medium containing no GM-CSF and counted, and plated at 5,000 cells/well (100 ⁇ l). A number.
  • the IL6 protein was diluted to 0.4 ⁇ g/ml with basal medium, 50 ⁇ l per well, and a protein content of 20 ng per well.
  • the antibody was diluted on another plate, starting at 100 ⁇ g/ml, and diluted 10 times with 10 gradients. Finally, 50 ⁇ l was added to the wells. Incubate for 7-10 days at 37 ° C and 5% CO 2 .
  • the surface of the chip was equilibrated with HBS-EP buffer at a flow rate of 10 ⁇ l/min for 5 min, and then the 1:1 mixture of "NHS + EDC" was injected at a flow rate of 10 ⁇ l/min for 7 min to activate the chip, which was diluted in 10 mM sodium acetate buffer.
  • the capture antibody Goat anti-mouse IgG
  • ethanolamine was injected at a flow rate of 10 ⁇ l/min for 7 min for surface blocking.
  • HBS-EP buffer Three pre-cycles were performed with HBS-EP buffer as a sample to balance the chip to stabilize the baseline, and the antibody diluted in HBS-EP buffer was injected at a flow rate of 10 ⁇ l/min for 0-5 min (the antibody and antigen-binding signal were controlled by adjusting the capture time).
  • the buffer was equilibrated for 1 min.
  • the low concentration antigen 0.33nM IL6 was injected at a flow rate of 30 ⁇ l/min for 5min, and the antigen was bound to the antibody.
  • 30 ⁇ l/min flow injection buffer for 15min the solution was dissociated at a flow rate of 100 ⁇ l/min for 50 times, and regenerated once every 10s. The loop ends.
  • the antigen concentration (eg, 1n IL6) was changed for the next gradient concentration cycle until all gradient concentrations (0.33 nM, 1 nM, 3 nM, 9 nM, 27 nM IL6) and repeated concentrations (eg, 9 nM IL6) were measured.

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Abstract

本发明提供了抗人IL6单克隆抗体、其重链可变区和轻链可变区的氨基酸序列，以及它们的编码核苷酸序列。本发明还提供了该抗人IL6单克隆抗体的制备方法和在制备抗肿瘤药物方面的用途。本发明提供的抗人IL6单克隆抗体可通过阻断IL6信号通路来抑制细胞增殖，进而实现肿瘤免疫治疗的目的。

Description

抗人IL6单克隆抗体及其制备方法和用途 技术领域
本发明属于肿瘤免疫疗法和分子免疫学领域，尤其是涉及抗人IL6的单克隆抗体。本发明还涉及该抗人IL6单克隆抗体的制备方法以及它们的用途。
背景技术
免疫系统是一种宿主防御系统，为了正常地发挥作用，免疫系统必须能够灵敏的检测到外来病原体的入侵，并将它们与生物体自身的健康组织区分开来。脊椎动物的免疫系统是一个由多种器官、组织、细胞和分子组成的功能性系统，是机体防卫外源物入侵的最有效的机制，这些免疫器官、组织、细胞和分子相互协作，相互制衡，从而达到保护机体免受外来侵染和维持体内平衡的作用(Janeway et al.,Immunology:The Immune System in Health and Disease.New York:Garland Science,2005)。这种相互协作、相互制衡需要众多免疫检查点蛋白和细胞因子的协调，刺激性免疫检查点蛋白增强免疫系统的防御反应，抑制性免疫检查点蛋白控制过强的免疫系统，防止产生自身免疫反应。癌变是正常体细胞失去正常细胞调节功能，基因突变累计到一定程度的结果。在肿瘤治疗中，肿瘤免疫治疗被广泛运用，它已成为肿瘤治疗的一个最重要的手段。肿瘤免疫治疗通过增强人体的免疫功能，使人体T细胞能更好的识别癌变细胞，从而起到杀伤癌细胞的作用。例如，针对抑制性免疫检查点蛋白PD1的抗体Pembrolizumab和针对CTLA4的Ipilimumab抗体已经被批准治疗多种癌症适应症。然而，由于肿瘤异质性和肿瘤免疫间的肿瘤间差异，这些基于免疫检查点蛋白的抗体药物临床益处还非常有限。
细胞因子里的最主要类别白细胞介素(Interleukins)具有引导免疫系统细胞成熟、分化、迁移和粘附的多种免疫调节功能。在肿瘤发生中，这些细胞因子直接刺激肿瘤部位的免疫效应物和基质细胞，并增强细胞毒性效应细胞的肿瘤细胞识别(Yoshimoto et al.,Immunotherapy 5:825-844,2009)。最近的一些研究表明白细胞介素参与许多肿瘤相关的分子机制，并且已被利用来开发许多基于细胞因子的癌症治疗方法。然而，肿瘤细胞有多种方法进行免疫逃逸，包括创建安全的肿瘤微环境、分泌抑制免疫系统的 细胞因子和抗炎性细胞因子、以及招募或转化抑制免疫反应的炎性细胞，包括调节性T(Treg)细胞、骨髓源性抑制细胞(MDSC)和树突状细胞细胞(DC)(Stewart et al.,Cancer Metastasis Rev.30:125-140,2011)。白细胞介素家族成员存在于肿瘤微环境中与各种生物分子如癌症干细胞、微小RNA、上皮间充质细胞相互作用。因此，能够基于白细胞介素影响肿瘤形成机制的原理，开发出高效的癌症免疫疗法。在过去十几年中，有一些包括白细胞介素2(IL2)、白细胞介素6(IL6)、白细胞介素7(IL7)、白细胞介素12(IL12)、白细胞介素18(IL18)和白细胞介素21(IL21)的相关疗法已进入患者临床试验阶段。
白细胞介素6(IL6)在体内有多种功能，其中该因子可以导致调节性T细胞(Treg)失衡并分泌多种炎症物质。研究表明患者血清IL6浓度升高与各种癌症(例如多发性骨髓瘤，非小细胞肺癌，结肠直肠癌，肾细胞癌，前列腺癌，乳腺癌和卵巢癌)的肿瘤分期和患者生存紧密相关。并且，IL6能够通过在乳腺癌细胞中诱导上皮间质细胞转换产生具有干细胞特质的CD44阳性细胞。有证据表明肿瘤相关内皮细胞中IL6的水平与肿瘤干细胞的致瘤性直接相关(Int.J.Mol.Sci.16:1691-1710,2015)。因此，阻断IL6信号传导是癌症的潜在治疗策略。强生制药的抗人IL6的Siltuximab抗体在2014年被美国药监局批准治疗Castleman病，并正在进行针对多发性骨髓瘤的临床二期实验。但迄今为止，还没有抗人IL6的抗体上市用于肿瘤免疫疗法。而且不同抗人IL6单克隆抗体也存在不同程度的副反应，包括可在某些患者中诱导免疫原性，以及不同IL6单克隆抗体具有不同程度的可开发性。因此，尚需开发新的能够阻断IL6信号的功能性抗体，其应具有更高的亲和力、特异性、功能性以及多样性。
发明内容
在一方面，本发明提供一种抗人IL-6单克隆抗体，其包括重链可变区和轻链可变区，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下组合之一：
(a)HCDR1的氨基酸序列为RYWMH；
HCDR2的氨基酸序列为YINPITGYTENNQKFKD；
HCDR3的氨基酸序列为GIRGFTY；
LCDR1的氨基酸序列为RASENVDNSDNSFMH；
LCDR2的氨基酸序列为RASNLDS；
LCDR3的氨基酸序列为QQTNEAPLT；
(b)HCDR1的氨基酸序列为NYWMH；
HCDR2的氨基酸序列为YVIPSTGYTDYNQSFKD；
HCDR3的氨基酸序列为LLPGFAY；
LCDR1的氨基酸序列为RSSQSLVDSNGNTYLH；
LCDR2的氨基酸序列为KVSNRFS；
LCDR3的氨基酸序列为SQSTHVPPT；
(c)HCDR1的氨基酸序列为NYWMH；
HCDR2的氨基酸序列为YIDPRTASIYYNQKFKD；
HCDR3的氨基酸序列为ILYGKYDV；
LCDR1的氨基酸序列为RSSQSLVDSNGNTYLH；
LCDR2的氨基酸序列为KVSNRFS；
LCDR3的氨基酸序列为SQSTHVPPT；
(d)HCDR1的氨基酸序列为DAWMD；
HCDR2的氨基酸序列为EIRSKTYHPATYYTKSVRG；
HCDR3的氨基酸序列为PRYYGGYFDY；
LCDR1的氨基酸序列为RASESVDNYGMSFMN；
LCDR2的氨基酸序列为TASNQGS；
LCDR3的氨基酸序列为QQSKEVPYT；
(e)HCDR1的氨基酸序列为NYIIH；
HCDR2的氨基酸序列为AIYPGNGDTSYSQKFKD；
HCDR3的氨基酸序列为GDAGYSAWFAY；
LCDR1的氨基酸序列为SASESVDSYGNNFMH；
LCDR2的氨基酸序列为LASKLES；
LCDR3的氨基酸序列为QQNNEDPLT；
(f)HCDR1的氨基酸序列为SHTVS；
HCDR2的氨基酸序列为KMWSNGDTDYDSAIRS；
HCDR3的氨基酸序列为YYFSSYGGGYFDY；
LCDR1的氨基酸序列为RASKSVSTYMH；
LCDR2的氨基酸序列为SASNLES；
LCDR3的氨基酸序列为QQSDELPDT；以及
(g)HCDR1的氨基酸序列为SFPMA；
HCDR2的氨基酸序列为TISPSGGTSYSRDSVKG；
HCDR3的氨基酸序列为ERIYNTYFDY；
LCDR1的氨基酸序列为LPSEDISSDLA；
LCDR2的氨基酸序列为NANTLPN；
LCDR3的氨基酸序列为QQYDSYPYT。
在更具体的实施方案中，所述抗人IL-6单克隆抗体的重链可变区和轻链可变区的氨基酸序列选自如下组合之一：
所述重链可变区包括如SEQ ID NO：1所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：3所示的氨基酸序列；
所述重链可变区包括如SEQ ID NO：5所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：7所示的氨基酸序列；
所述重链可变区包括如SEQ ID NO：9所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：11所示的氨基酸序列；
所述重链可变区包括如SEQ ID NO：13所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：15所示的氨基酸序列；
所述重链可变区包括如SEQ ID NO：17所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：19所示的氨基酸序列；
所述重链可变区包括如SEQ ID NO：21所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：23所示的氨基酸序列；以及
所述重链可变区包括如SEQ ID NO：25所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：27所示的氨基酸序列。
在优选的实施方案中，所述抗人IL6单克隆抗体与IL6之间的解离常数KD小于10nM。在更优选的实施方案中，所述抗人IL6单克隆抗体与IL6之间的解离常数KD小于1nM。
在一个实施方案中，所述抗人IL6单克隆抗体抑制IL6对TF-1细胞的增殖促进作用。
在另一方面，本发明提供了分离的多核苷酸，其编码所述抗人IL6单克隆抗体。
在一个具体实施方案中，所述多核苷酸包括编码所述抗人IL6单克隆抗体的重链可变区的重链可变区编码序列和编码所述抗人IL6单克隆抗体的轻链可变区的轻链可变区编码序列，所述重链可变区编码序列和轻链可变区编码序列选自如下组合之一：
所述重链可变区编码序列包括如SEQ ID NO：2所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：4所示的核苷酸序列；
所述重链可变区编码序列包括如SEQ ID NO：6所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：8所示的核苷酸序列；
所述重链可变区编码序列包括如SEQ ID NO：10所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：12所示的核苷酸序列；
所述重链可变区编码序列包括如SEQ ID NO：14所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：16所示的核苷酸序列；
所述重链可变区编码序列包括如SEQ ID NO：18所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：20所示的核苷酸序列；
所述重链可变区编码序列包括如SEQ ID NO：22所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：24所示的核苷酸序列；以及
所述重链可变区编码序列包括如SEQ ID NO：26所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：28所示的核苷酸序列。
在另一方面，本发明提供了包括上述多核苷酸的表达载体。
在另一方面，本发明提供了包括上述表达载体的宿主细胞。
在优选的实施方案中，所述宿主细胞为HEK293-6E细胞。
在另一方面，本发明提供了一种制备抗人IL6单克隆抗体的方法，包括以上述表达载体转染感受态细胞，并对所述细胞进行培养。
在另一方面，本发明提供了所述抗人IL6单克隆抗体、所述多核苷酸、所述表达载体、所述宿主细胞在制备用于抗肿瘤的药物中的用途。
在优选的实施方案中，所述肿瘤选自多发性骨髓瘤、非小细胞肺癌、结肠直肠癌、肾细胞癌、前列腺癌、乳腺癌和卵巢癌。
在另一方面，本发明提供了抗肿瘤药物组合物，其包含有效量的所述抗人IL6单克隆抗体和药学上可接受的载体。
在又一方面，本发明提供了一种制备抗人IL6单克隆抗体的方法，包括：
1)以人IL6免疫小鼠，在所述小鼠中产生针对人IL6的免疫反应；
2)取所述小鼠的脾脏细胞与骨髓瘤细胞融合，并对获得的杂交瘤细胞进行筛选，得到特异识别人IL6的阳性母克隆；
3)对所述阳性母克隆进行亚克隆，以获得稳定的杂交瘤细胞株；
4)对所述杂交瘤细胞株进行基因测序，获得抗人IL6抗体的轻链和重链的可变区编码序列；以及
5)用所述可变区编码序列进行重组抗体生产，获得功能性抗人IL6单克隆抗体。
本发明提供的抗IL6单克隆抗体对IL6具有高亲和性、高特异性、并且能够分别识别IL6的不同表位，能够刺激或抑制IL6下游通路，激活或抑制T细胞分泌细胞因子，例如通过抑制IL6下游通路来抑制TF-1细胞增值。因而，本发明提供的针对IL6的功能性单克隆抗体，可通过阻断IL6信号通路来抑制细胞增殖，进而实现肿瘤免疫治疗的目的。
附图说明
图1显示了经人IL6免疫后的小鼠的血清ELISA效价检测结果。
图2显示了经人IL6免疫后的大鼠的血清ELISA效价检测结果。
图3显示了本发明的抗IL6单克隆抗体与IL6重组蛋白特异性结合的曲线图。
图4显示了本发明的抗IL6单克隆抗体特异性抑制TF-1细胞增殖的曲线图。
图5显示了本发明的抗IL6单克隆抗体对IL6蛋白的亲和力的测定结果。
具体实施方式
除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。
本文所用的术语“抗体”指免疫球蛋白分子，其通常为由2个相同重链和2个相同轻链通过二硫键相互连接组成的四聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。在重链和轻链的可变区内，分别有三个局部区域的氨基酸组成和排列顺序具有更高的变异程度，为抗体与抗 原结合的关键位置，因而也称为互补决定区(CDR)。在本文中，三个重链互补决定区分别称为HCDR1、HCDR2和HCDR3，三个轻链互补决定区分别称为LCDR1、LCDR2和LCDR3。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。根据它们重链恒定区的氨基酸序列，可将抗体分为不同类别。有五种主要类型的完整抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可进一步分为亚类，例如，IgG1、IgG2、IgG3、IgG4、IgA和IgA2。不同类别的免疫球蛋白的亚单位结构和三维构象在本领域内是已知的。本发明旨在包括任何前述类或亚类的抗体。
本文使用的术语“抗体”还旨在涵盖其消化片段或功能性变体，例如，能够结合IL6或其部分的抗体片段，包括但不限于Fab(例如，抗体经木瓜蛋白酶消化而得到)、F(ab’)2(例如，通过胃蛋白酶消化得到)、Fv或scFv(例如通过分子生物学技术得到)。
本文使用的术语“单克隆抗体”指均一的、仅针对某一特定抗原表位的抗体。与典型地包括针对不同抗原决定簇(表位)的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体优选通过重组DNA方法产生，或通过本文其它地方描述的筛选方法获得。
本文使用的术语“分离的多核苷酸”指非自然界中天然存在状态的多核苷酸，包括通过生物学技术从自然界(包括生物体内)分离出的多核苷酸，也包括人工合成的多核苷酸。分离的多核苷酸可以是基因组DNA、cDNA、mRNA或合成的其它RNA，或者它们的组合。本文提供了多个用于编码抗人IL6单克隆抗体的重链可变区和轻链可变区的核苷酸序列，需要指出的是，本领域技术人员可以根据本文所提供的重链可变区和轻链可变区的氨基酸序列，基于密码子简并性，设计出与以上提供的核苷酸序列不完全相同的核苷酸序列，但都编码相同的氨基酸序列。这些经改动的核苷酸序列也包括在本发明的范围内。
当涉及多核苷酸时，本文所用的术语“载体”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如，核酸、质粒、或病毒等)。术语“表达载体”或“表达盒”指适于在宿主细胞内表达目的基因(待表达核苷酸序列)的载体，通常包括目的基因、启动子、终止子、标记基因等部分。
本文所用的术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态 或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
本文所用的术语“转染”指外来或外源DNA被细胞摄入，该技术可用于将一种或多种外源DNA部分导入适宜的宿主细胞。可通过理化方法(例如通过氯化钙处理)诱导细胞，使其处于最适摄取和容纳外来DNA的生理状态，即“感受态”。
提及药物组合物时，本文所使用的术语“有效量的”指可对人和/或动物产生功能或活性且可被人和/或动物所接受的量。“药学上可接受的载体”指用于给药的载体，包括各种赋形剂、稀释剂和缓冲剂等，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。
下面将结合具体实施例对本发明的一些方面进行详细描述。除非另有说明，下文描述的实施例的方法和材料均为可以通过市场购买获得的常规产品。
实施例1：人IL6杂交瘤细胞株的获得
1)动物免疫
抗原采用tag-free的人IL6重组蛋白(GenScript，Cat.No.Z03034)。用含50μg IL6蛋白的200μl弗氏完全佐剂(Sigma-Aldrich)的1:1乳液皮下免疫雌性Balb/c和C57bl/6小鼠以及Wistar大鼠。随后，每二周腹腔/皮下交替注射含25μg IL6的弗氏不完全佐剂(Sigma-Aldrich)的1:1乳液最多达3次，从而对小鼠进行加强免疫。骨髓瘤融合前4天，对表现出最高抗体滴度(参见图1和图2，采用血清ELISA法测定抗体效价)的一只小鼠(#3509)和一只大鼠(#3693)进行了25μg IL6(不含佐剂)腹腔加强免疫。
2)杂交瘤融合和筛选
提取脾脏并进行均质化以产生单细胞悬液，同时准备骨髓瘤细胞(SP2/0)单细胞悬液。使用电融合将8.9×10 7个脾细胞与4.1×10 7个SP2/0小鼠骨髓瘤细胞进行融合。将融合的细胞重悬于100ml含杂交瘤细胞选择剂胸腺核苷嘧啶、次黄嘌呤和氨基喋呤的DMEM/10％FBS培养基中，并用移液管以100μl的体积移至96孔板中。将板在37℃下在6％CO 2中孵育。孵育7天之后，开始使用下文所述的ELISA结合来测试针对IL6的抗体的存在情况。
ELISA结合检测方法：将间接ELISA用于评估上清液中抗体对于IL6的结合能 力。将ELISA板(Nunc)用100μl/孔的PBS中0.5μg/ml的重组IL6蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用200μl/孔的含1％BSA的PBST在37℃封闭0.5小时。随后弃去封闭液，向每个板加入100μl杂交瘤细胞培养上清液，然后在室温下孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗小鼠IgG(Fab-特异性)(GenScript)37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液(GenScript)并在室温下在黑暗中孵育15分钟。通过加入50μl的1MHCl终止液(Sigma)终止反应。使用酶标仪在450nm下读板。
3)杂交瘤亚克隆
使用有限稀释法进行亚克隆。使用血球细胞计数器并在含杂交瘤细胞选择剂胸腺核苷嘧啶、次黄嘌呤和氨基喋呤的DMEM/10％FBS培养基中对细胞进行系列稀释来确定细胞数量，直至细胞密度达到5-15个细胞/ml。对于每个杂交瘤，将200μl的细胞溶液用移液管移至96孔中，密度为1-3个细胞/孔。将培养物在37℃下在5％CO 2中培养1周后，对上清液进行上述ELISA结合测试，来评估针对IL6的抗体的存在情况。
实施例2：单克隆抗体的可变区测序及抗体重组生产
使用快速ELISA小鼠抗体亚型鉴定试剂盒(Clonotyping System-HRP，SouthernBiotech)对杂交瘤细胞培养上清中的抗体进行亚型鉴定后，使用TRIzol(Ambion)从3×10 6-5×10 6个杂交瘤细胞提取总RNA，并利用抗体亚型特异性引物和通用引物(PrimeScript TM 1stStrand cDNA Synthesis Kit，Takara)将其逆转录为cDNA。随后通过RACE PCR(GenScript)扩增鼠免疫球蛋白重链和轻链V-区域片段，将所得的PCR片段亚克隆至pMD18-T载体系统(Takara)中，并使用载体特异性引物对插入片段进行测序。最终获得了克隆8H10B7E6、23A9H3、29D6B5、41A10B8、49F10H6、53A2F9(大鼠)、61G1D8(大鼠)的独特V-区域核苷酸/氨基酸序列。在重链和轻链的氨基酸序列中，CDR1区加有实心下划线，CDR2区加有虚线下划线，CDR3区加有波浪下划线。
8H10B7E6重链可变区氨基酸序列：SEQ ID NO:1
Figure PCTCN2018119353-appb-000001
8H10B7E6重链可变区核苷酸序列：SEQ ID NO:2
Figure PCTCN2018119353-appb-000002
8H10B7E6轻链可变区氨基酸序列：SEQ ID NO:3
Figure PCTCN2018119353-appb-000003
8H10B7E6轻链可变区核苷酸序列：SEQ ID NO:4
Figure PCTCN2018119353-appb-000004
23A9H3重链可变区氨基酸序列：SEQ ID NO:5
Figure PCTCN2018119353-appb-000005
23A9H3重链可变区核苷酸序列：SEQ ID NO:6
Figure PCTCN2018119353-appb-000006
23A9H3轻链可变区氨基酸序列：SEQ ID NO:7
Figure PCTCN2018119353-appb-000007
Figure PCTCN2018119353-appb-000008
23A9H3轻链可变区核苷酸序列：SEQ ID NO:8
Figure PCTCN2018119353-appb-000009
29D6B5重链可变区氨基酸序列：SEQ ID NO:9
Figure PCTCN2018119353-appb-000010
29D6B5重链可变区核苷酸序列：SEQ ID NO:10
Figure PCTCN2018119353-appb-000011
29D6B5轻链可变区氨基酸序列：SEQ ID NO:11
Figure PCTCN2018119353-appb-000012
29D6B5轻链可变区核苷酸序列：SEQ ID NO:12
Figure PCTCN2018119353-appb-000013
41A10B8重链可变区氨基酸序列：SEQ ID NO:13
Figure PCTCN2018119353-appb-000014
41A10B8重链可变区核苷酸序列：SEQ ID NO:14
Figure PCTCN2018119353-appb-000015
41A10B8轻链可变区氨基酸序列：SEQ ID NO:15
Figure PCTCN2018119353-appb-000016
41A10B8轻链可变区核苷酸序列：SEQ ID NO:16
Figure PCTCN2018119353-appb-000017
49F10H6重链可变区氨基酸序列：SEQ ID NO:17
Figure PCTCN2018119353-appb-000018
49F10H6重链可变区核苷酸序列：SEQ ID NO:18
Figure PCTCN2018119353-appb-000019
Figure PCTCN2018119353-appb-000020
49F10H6轻链可变区氨基酸序列：SEQ ID NO:19
Figure PCTCN2018119353-appb-000021
49F10H6轻链可变区核苷酸序列：SEQ ID NO:20
Figure PCTCN2018119353-appb-000022
53A2F9重链可变区氨基酸序列：SEQ ID NO:21
Figure PCTCN2018119353-appb-000023
53A2F9重链可变区核苷酸序列：SEQ ID NO:22
Figure PCTCN2018119353-appb-000024
53A2F9轻链可变区氨基酸序列：SEQ ID NO:23
Figure PCTCN2018119353-appb-000025
53A2F9轻链可变区核苷酸序列：SEQ ID NO:24
Figure PCTCN2018119353-appb-000026
Figure PCTCN2018119353-appb-000027
61G1D8重链可变区氨基酸序列：SEQ ID NO:25
Figure PCTCN2018119353-appb-000028
61G1D8重链可变区核苷酸序列：SEQ ID NO:26
Figure PCTCN2018119353-appb-000029
61G1D8轻链可变区氨基酸序列：SEQ ID NO:27
Figure PCTCN2018119353-appb-000030
61G1D8轻链可变区核苷酸序列：SEQ ID NO:28
Figure PCTCN2018119353-appb-000031
分别合成包含轻链可变区+恒定区与重链可变区+恒定区的DNA片段，将其分别插入pTT5表达载体中，形成表达质粒。
将上述表达质粒共转染HEK293-6E细胞，并于37℃摇瓶中培养10天后，收取上清用于抗体纯化。纯化之前，将管道和蛋白A柱用0.2M NaOH去热原。将柱用含有0.05M Tris和1.5M NaCl(pH8.0)的缓冲液重新平衡。随后将收获的细胞培养物上清 液，使用2×上述缓冲液1:1稀释并过滤除菌。将过滤的上清液和蛋白A柱室温孵育2小时，用并1×上述缓冲液洗涤柱后，使用无菌0.1M柠檬酸钠(pH3.5)洗脱IgG，收集了洗脱液并用九分之一体积的无菌1M Tris-HCl(pH9)中和。在无菌条件下，将所述产品缓冲液交换为PBS(pH7.4)以除去任何的洗脱缓冲液并浓缩所述样品。浓缩之后，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量。
纯化的抗体通过BioRad电泳系统用10％预制胶(GenScript)通过SDS-PAGE来分析。将所述凝胶用Estain2.0(GenScript)染色并通过比较染色带与Protein Ladder(GenScript)来估计分子大小和纯度。
实施例3：单克隆抗体对人IL6重组蛋白的结合
将间接ELISA用于评估纯化抗体对于IL6的结合能力。将ELISA板(Nunc)用100μl/孔的PBS中0.5μg/ml的重组IL6蛋白在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用200μl/孔的含1％BSA的PBST在37℃封闭0.5小时。随后弃去封闭液，向首孔加入10μg/ml的纯化抗体100μl，并按照3倍梯度稀释，共计11个测试浓度梯度。然后在室温下孵育1小时。将板用PBST洗涤三次，并用100μl/孔的缀合辣根过氧化物酶的山羊抗小鼠/大鼠IgG(Fab-特异性)(GenScript)37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液(GenScript)并在室温下在黑暗中孵育15分钟。通过加入50μl的1M HCl终止液(Sigma)终止反应。使用酶标仪在450nm下读板。克隆8H10B7E6、23A9H3、29D6B5、41A10B8、49F10H6、53A2F9(大鼠)、61G1D8(大鼠)对于重组蛋白IL6的结合能力如图3。根据ELISA浓度梯度实验计算得到的EC50表明这些抗体对于抗原都表现出相当高的亲和力(ELISA EC50都小于25ng/ml；特别是53A2F9达到6.32ng/ml，相当于0.04nM)。
实施例4：单克隆抗体表位鉴定
将竞争ELISA用于评估纯化抗体的抗原表位。将ELISA板(Nunc)用100μl/孔的PBS中0.5μg/ml的重组IL6在4℃下包被过夜。用PBS-T(0.05％吐温)洗涤板，并将其用200μl/孔的含1％BSA的PBST在37℃封闭0.5小时。随后弃去封闭液，每孔分别加入一对(其中一个已标记生物素)用于竞争实验的待测抗体，每个纯化抗体100μl(10μg/ml)。然后在37℃下孵育1小时。将板用PBST洗涤3次，并用100μl/孔的抗生物素蛋白链菌素HRP(SA-HRP，GenScript)37℃孵育10分钟。将板用PBST洗 涤五次，然后加入TMB显色液(GenScript)并在室温下在黑暗中孵育15分钟。通过加入50μl的1MHCl终止液(Sigma)终止反应。使用酶标仪在450nm下读板。测定结果显示，克隆8H10B7E6、23A9H3、29D6B5是针对同一个表位，而41A10B8、49F10H6、53A2F9(大鼠)、和61G1D8(大鼠)分别针对另外的四个不同表位。这些结果表明所得到的特异性针对IL6抗体具有多个不同的抗原识别表位。因此，本发明筛选到的抗体具有更大的多样性。
实施例5：单克隆抗体的功能性检测
TF-1细胞的生长是粒细胞巨噬细胞集落刺激因子(GM-CSF)依赖的，如果该因子缺乏或功能被阻碍，细胞的增殖、分化、成熟会受到明显的抑制。本单克隆抗体功能实验是将培养基中的GM-CSF替换成有同样功能的IL6蛋白，再通过加入抗IL6单克隆抗体，通过判断培养一定时间后的TF-1活细胞的数量来确定该单克隆抗体是否能阻断IL-6蛋白生物学功能。在含有GM-CSF的培养基中正常培养TF-1细胞，实验前将细胞收集，用不含有GM-CSF的基础培养基洗涤2次并计数，按5000个/孔(100μl)铺96孔板若干。IL6蛋白用基础培养基稀释至0.4μg/ml，每孔添加50μl，每孔蛋白含量为20ng。抗体在另外一块板上，起始100μg/ml稀释，2倍稀释10个梯度。最终向板孔内加50μl。37℃及5％CO 2条件下孵育7-10天。每孔加20μl Promega Substrate Cell Titer 96Aqueous One Solution Reagent。37度孵育，每半小时用490nm读数一次，挑选线性最好的编辑数据。IL6抗体对TF-1细胞生长抑制作用见图4。这些结果表明，本发明筛选到的抗体能够有效的抑制细胞增殖，特别是克隆53A2F9和61G1D8抗体的抑制IC50分别达到3.95μg/ml，和4.54μg/ml。
实施例6：单克隆抗体的亲和力测定
以10μl/min流速的HBS-EP缓冲液平衡芯片表面5min，随后以10μl/min的流速注射“NHS+EDC”的1：1混合液7min来活化芯片，将稀释在10mM醋酸钠缓冲液中的捕获抗体(Goat anti-mouse IgG)以10μl/min流速注射约7min进行耦联，最后以10μl/min的流速注射乙醇胺7min进行表面封闭。
以HBS-EP缓冲液作为样品进行三个预循环来平衡芯片使基线稳定，10μl/min流速注射稀释在HBS-EP缓冲液中的抗体0～5min(通过调整捕获时间来控制抗体和抗原结合信号在～100RU)，缓冲液平衡1min。以30μl/min流速注射低浓度抗原0.33nM  IL6 5min，抗原与抗体发生结合，之后30μl/min流速注射缓冲液15min进行解离，100μl/min流速注射50mMHCl共四次，每次10s进行再生，一次循环结束。改变抗原浓度(如1n IL6)进行下一个梯度浓度的循环测定直到所有梯度浓度(0.33nM、1nM、3nM、9nM、27nM IL6)及重复浓度(如9nM IL6)测定结束。
实验数据经过双扣减(对照通道及零浓度)后，在Biacore 8K evaluation software中进行“1:1Binding”模型的拟合。使用Biacore 8K测定抗体针对IL重组蛋白的亲和力。如图5所示，特异性针对IL6的单克隆抗体对IL6的亲和力由Biacore测得均达到sub-nM级至pM级。这些结果表明，本发明筛选到的抗体具有非常高的亲和力。
本发明所属领域技术员应理解，以上描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。

Claims (15)

  1. 抗人IL-6单克隆抗体，其包括重链可变区和轻链可变区，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3选自如下组合之一：
    (a)HCDR1的氨基酸序列为RYWMH；
    HCDR2的氨基酸序列为YINPITGYTENNQKFKD；
    HCDR3的氨基酸序列为GIRGFTY；
    LCDR1的氨基酸序列为RASENVDNSDNSFMH；
    LCDR2的氨基酸序列为RASNLDS；
    LCDR3的氨基酸序列为QQTNEAPLT；
    (b)HCDR1的氨基酸序列为NYWMH；
    HCDR2的氨基酸序列为YVIPSTGYTDYNQSFKD；
    HCDR3的氨基酸序列为LLPGFAY；
    LCDR1的氨基酸序列为RSSQSLVDSNGNTYLH；
    LCDR2的氨基酸序列为KVSNRFS；
    LCDR3的氨基酸序列为SQSTHVPPT；
    (c)HCDR1的氨基酸序列为NYWMH；
    HCDR2的氨基酸序列为YIDPRTASIYYNQKFKD；
    HCDR3的氨基酸序列为ILYGKYDV；
    LCDR1的氨基酸序列为RSSQSLVDSNGNTYLH；
    LCDR2的氨基酸序列为KVSNRFS；
    LCDR3的氨基酸序列为SQSTHVPPT；
    (d)HCDR1的氨基酸序列为DAWMD；
    HCDR2的氨基酸序列为EIRSKTYHPATYYTKSVRG；
    HCDR3的氨基酸序列为PRYYGGYFDY；
    LCDR1的氨基酸序列为RASESVDNYGMSFMN；
    LCDR2的氨基酸序列为TASNQGS；
    LCDR3的氨基酸序列为QQSKEVPYT；
    (e)HCDR1的氨基酸序列为NYIIH；
    HCDR2的氨基酸序列为AIYPGNGDTSYSQKFKD；
    HCDR3的氨基酸序列为GDAGYSAWFAY；
    LCDR1的氨基酸序列为SASESVDSYGNNFMH；
    LCDR2的氨基酸序列为LASKLES；
    LCDR3的氨基酸序列为QQNNEDPLT；
    (f)HCDR1的氨基酸序列为SHTVS；
    HCDR2的氨基酸序列为KMWSNGDTDYDSAIRS；
    HCDR3的氨基酸序列为YYFSSYGGGYFDY；
    LCDR1的氨基酸序列为RASKSVSTYMH；
    LCDR2的氨基酸序列为SASNLES；
    LCDR3的氨基酸序列为QQSDELPDT；以及
    (g)HCDR1的氨基酸序列为SFPMA；
    HCDR2的氨基酸序列为TISPSGGTSYSRDSVKG；
    HCDR3的氨基酸序列为ERIYNTYFDY；
    LCDR1的氨基酸序列为LPSEDISSDLA；
    LCDR2的氨基酸序列为NANTLPN；
    LCDR3的氨基酸序列为QQYDSYPYT。
  2. 如权利要求1所述的抗人IL6单克隆抗体，其重链可变区和轻链可变区的氨基酸序列选自如下组合之一：
    所述重链可变区包括如SEQ ID NO：1所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：3所示的氨基酸序列；
    所述重链可变区包括如SEQ ID NO：5所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：7所示的氨基酸序列；
    所述重链可变区包括如SEQ ID NO：9所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：11所示的氨基酸序列；
    所述重链可变区包括如SEQ ID NO：13所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：15所示的氨基酸序列；
    所述重链可变区包括如SEQ ID NO：17所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：19所示的氨基酸序列；
    所述重链可变区包括如SEQ ID NO：21所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：23所示的氨基酸序列；以及
    所述重链可变区包括如SEQ ID NO：25所示的氨基酸序列，所述轻链可变区包括如SEQ ID NO：27所示的氨基酸序列。
  3. 如权利要求1或2所述的抗人IL6单克隆抗体，其与IL6之间的解离常数KD小于10nM。
  4. 如权利要求1或2所述的抗人IL6单克隆抗体，其与IL6之间的解离常数KD小于1nM。
  5. 如权利要求1或2所述的抗人IL6单克隆抗体，其抑制IL6对TF-1细胞的增殖促进作用。
  6. 分离的多核苷酸，其编码权利要求1或2所述的抗人IL6单克隆抗体。
  7. 如权利要求6所述的多核苷酸，其包括编码所述抗人IL6单克隆抗体的重链可变区的重链可变区编码序列和编码所述抗人IL6单克隆抗体的轻链可变区的轻链可变区编码序列，所述重链可变区编码序列和轻链可变区编码序列选自如下组合之一：
    所述重链可变区编码序列包括如SEQ ID NO：2所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：4所示的核苷酸序列；
    所述重链可变区编码序列包括如SEQ ID NO：6所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：8所示的核苷酸序列；
    所述重链可变区编码序列包括如SEQ ID NO：10所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：12所示的核苷酸序列；
    所述重链可变区编码序列包括如SEQ ID NO：14所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：16所示的核苷酸序列；
    所述重链可变区编码序列包括如SEQ ID NO：18所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：20所示的核苷酸序列；
    所述重链可变区编码序列包括如SEQ ID NO：22所示的核苷酸序列，所述轻链可变区编码序列包括如SEQ IDNO：24所示的核苷酸序列；以及
    所述重链可变区编码序列包括如SEQ ID NO：26所示的核苷酸序列，所述轻链可 变区编码序列包括如SEQ IDNO：28所示的核苷酸序列。
  8. 包括权利要求6或7所述的多核苷酸的表达载体。
  9. 包括权利要求8所述的表达载体的宿主细胞。
  10. 如权利要求8所述的宿主细胞，其为HEK293-6E细胞。
  11. 一种制备抗人IL6单克隆抗体的方法，包括以权利要求8所述的表达载体转染感受态细胞，并对所述细胞进行培养。
  12. 权利要求1至5中任一项所述的抗人IL6单克隆抗体、权利要求6或7所述的多核苷酸、权利要求8所述的表达载体、权利要求9或10所述的宿主细胞在制备用于抗肿瘤的药物中的用途。
  13. 如权利要求12所述的用途，其中所述肿瘤选自多发性骨髓瘤、非小细胞肺癌、结肠直肠癌、肾细胞癌、前列腺癌、乳腺癌和卵巢癌。
  14. 抗肿瘤药物组合物，其包含有效量的权利要求1至5中任一项所述的抗人IL6单克隆抗体和药学上可接受的载体。
  15. 一种制备抗人IL6单克隆抗体的方法，包括
    1)以人IL6免疫小鼠，在所述小鼠中产生针对人IL6的免疫反应；
    2)取所述小鼠的脾脏细胞与骨髓瘤细胞融合，并对获得的杂交瘤细胞进行筛选，得到特异识别人IL6的阳性母克隆；
    3)对所述阳性母克隆进行亚克隆，以获得稳定的杂交瘤细胞株；
    4)对所述杂交瘤细胞株进行基因测序，获得抗人IL6抗体的轻链和重链的可变区编码序列；以及
    5)用所述可变区编码序列进行重组抗体生产，获得功能性抗人IL6单克隆抗体。
PCT/CN2018/119353 2017-12-05 2018-12-05 抗人il6单克隆抗体及其制备方法和用途 WO2019109947A1 (zh)

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