CN111057152B - Anti-human IL-6Ra monoclonal antibody and application thereof - Google Patents
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Abstract
The invention discloses an anti-human IL-6Ra monoclonal antibody and application thereof, the anti-human IL-6Ra monoclonal antibody comprises: a variable heavy chain, wherein the 3 Complementarity Determining Regions (CDRs) of the variable heavy chain are SEQ ID NOs: 1. SEQ ID NO: 2 and SEQ ID NO: 3; a variable light chain, wherein the 3 Complementarity Determining Regions (CDRs) of the variable heavy chain are SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6. the monoclonal antibody obtained by the invention not only prompts that the targeted IL-6/IL-6R axis can be used as an effective target spot for intervening inflammatory diseases, provides an important basis for exploring the treatment of systemic inflammatory response syndrome, but also carries out reconstruction and expression on the antibody to a plurality of small molecular genetic engineering antibodies, such as single chain antibodies, chimeric antibodies, Fab antibodies and the like, and can achieve the application of the drugs for treating, diagnosing or preventing Systemic Inflammatory Response Syndrome (SIRS), liver cancer and/or glioma.
Description
Technical Field
The invention belongs to the field of gene medicines, and relates to an anti-human IL-6Ra monoclonal antibody and application thereof.
Background
Systemic Inflammatory Response Syndrome (SIRS) is a Systemic inflammatory response in which the body is uncontrollably self-sustained amplified and self-destroyed due to the action of infectious or non-infectious causes on the body. When the body is struck by exogenous injury or infection of toxic substances, the initial inflammatory reaction can be promoted, and meanwhile, endogenous immune inflammatory factors generated by the body form a 'waterfall effect'. In critically ill patients, SIRS is most likely to be caused due to the reduction of compensatory anti-inflammatory response capability and metabolic dysfunction of the body, i.e., the disease mediators spill into plasma and cause systemic inflammation at distant sites, and serious patients can cause Multiple Organ Dysfunction Syndrome (MODS), which is an inflammatory response affecting the whole body and is also one of cytokine storms. How to find out ideal, efficient and specific medicines which can not only rapidly inhibit the systemic inflammatory reaction (body to various cytokines/inflammatory mediators), but also effectively and selectively block the pathological process of the body aiming at the pathogenic mechanism of the body becomes a key problem to be solved urgently in the research on the systemic inflammatory reaction syndrome.
Interleukin 6 receptor (IL-6R) is a type I transmembrane protein, cloned and described in 1988 (Yamasaki et al 1988) as a member of the hematopoietic cytokine receptor superfamily, whose genes are located on human chromosome 1q21.3, expressed in lymphocytes such as monocytes, macrophages, etc., and serves as a receptor for the multifunctional cytokine IL-6. Studies have shown that IL-6R is highly expressed in some leukemia cells in addition to multiple myeloma, plasmacytoma, Kaposi's sarcoma, and hepatoma cells. Obstruction of this pathway can lead to the development of acute and chronic inflammation, autoimmune diseases or tumors. Targeting the IL-6/IL-6R axis can serve as an effective target for intervention in a number of inflammatory or neoplastic diseases.
In 2017, the FDA announces that tolytin monoclonal antibodies (mabs) targeting human interleukin 6 receptor (IL-6R) are used for treating Cytokine Release Syndrome (CRS) caused by chimeric antigen receptor T (CAR-T) cell therapy, which is another important indication after the successful use of the mabs in autoimmune diseases such as Rheumatoid Arthritis (RA), Juvenile Idiopathic Arthritis (JIA), etc., and IL-6/IL-6R and targeted inhibitors thereof directed to the pathway control or reduce systemic inflammatory response through blocking of the signal, thereby also enlightening the search of new strategies for targeting the IL-6/IL-6R system for treating inflammatory diseases or tumors.
Disclosure of Invention
An object of the present invention is to provide a monoclonal antibody against human IL-6 Ra. The heavy chain and light chain variable region genes of the antibody are cloned and prepared from a mouse hybridoma cell strain IL-6Ra-C5 capable of secreting high activity (the preservation number is CCTCC NO: C2019126).
An anti-human IL-6Ra monoclonal antibody comprising:
a variable heavy chain, wherein the 3 Complementarity Determining Regions (CDRs) of the variable heavy chain are SEQ ID NOs: 1. SEQ ID NO: 2 and SEQ ID NO: 3;
a variable light chain, wherein the 3 Complementarity Determining Regions (CDRs) of the variable heavy chain are SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.
an anti-human IL-6Ra monoclonal antibody comprising:
a heavy chain variable region gene having the amino acid sequence of SEQ ID NO: 7;
a light chain variable region gene having the amino acid sequence of SEQ ID NO: 8, or a pharmaceutically acceptable salt thereof.
An anti-human IL-6Ra monoclonal antibody comprising: consisting of SEQ ID NO: 9 and the variable heavy chain encoded by SEQ ID NO: 10, a variable light chain.
Alternatively, the 3 Complementarity Determining Regions (CDRs) of the variable heavy chain are SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3;
the 3 Complementarity Determining Regions (CDRs) of the variable heavy chain are SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.
a polypeptide product encoded by said anti-human IL-6Ra monoclonal antibody.
Alternatively, the polypeptide product encoded by the heavy chain variable region gene has the amino acid sequence of SEQ ID NO: 9, and (c) a sequence of (c);
the polypeptide product coded by the light chain variable region gene has the sequence shown in SEQ ID NO: 10, or a fragment thereof.
A hybridoma cell, wherein the preservation number of the hybridoma cell is CCTCC C2019126.
An anti-human IL-6Ra monoclonal antibody produced by said hybridoma.
The anti-human IL-6Ra monoclonal antibody, wherein the monoclonal antibody is a murine antibody, a human antibody or a humanized antibody;
or the monoclonal antibody is a chimeric antibody, a small molecule antibody, a multivalent minibody, a bispecific antibody, a recombinant antibody fusion protein, a recombinant immunotoxin, or a phage antibody.
The anti-human IL-6Ra monoclonal antibody is applied to preparing medicines for treating, diagnosing or preventing Systemic Inflammatory Response Syndrome (SIRS), liver cancer and/or glioma.
A composition comprising said anti-human IL-6Ra monoclonal antibody.
Optionally, the composition is an antibody-nuclide conjugate, an antibody-chemotherapeutic drug conjugate, an antibody-toxin conjugate, an antibody-Biological Response Modifier (BRM) conjugate, an antibody-directed enzymatic prodrug, or an immunoliposome.
An anti-human IL-6Ra monoclonal antibody comprising:
a) SEQ ID NO: 4, CDR1 of the light chain variable region as set forth in the amino acid sequence of seq id no;
b) SEQ ID NO: 5, CDR2 of the light chain variable region as set forth in the amino acid sequence of seq id no;
c) SEQ ID NO: 6, CDR3 of the light chain variable region as set forth in the amino acid sequence of seq id no;
d) SEQ ID NO: 1, CDR1 of the heavy chain variable region as set forth in the amino acid sequence of seq id no;
e) SEQ ID NO: 2, CDR2 of the heavy chain variable region as set forth in the amino acid sequence of seq id no;
and f) SEQ ID NO: 3, CDR3 of the heavy chain variable region as set forth in the amino acid sequence of seq id no.
The kit comprises the anti-human IL-6Ra monoclonal antibody.
A kit, comprising:
a) said anti-human IL-6Ra monoclonal antibody, and b) a conjugate comprising an antibody linked to a signal producing compound, wherein the antibody in said conjugate is different from said anti-human IL-6Ra monoclonal antibody.
A kit, comprising: a) said anti-human IL-6Ra monoclonal antibody, and b) a conjugate comprising an antigen linked to a signal producing compound.
The invention also aims at the application of the antibody in the preparation of medicines for diagnosing and treating IL-6 Ra-related inflammatory diseases, such as the development and application in inhibiting the release of inflammatory cytokines. The result of the research, which adopts humanized IL-6Ra fixed point knock-in transgenic mice to prepare a systemic inflammatory response syndrome SIRS model, and uses anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab for evaluation shows that the antibody can obviously relieve LPS-induced IL-6Ra transgenic mouse SIRS symptoms, and the treatment of the antibody can obviously reduce the expression levels of four proinflammatory cytokines, namely TNF-alpha, IL-1 beta, INF-gamma and IL-6, in the blood serum of LPS injected mice.
The inventor successfully clones antibody light and heavy chain variable region genes from cultured anti-tumor specific monoclonal antibody IL6RA-C5Ab hybridoma cells by using a set of designed primers. Sequence analysis shows that the germ line gene of the antibody heavy chain variable region gene VH is derived from Musmus IGHV3-2 x 02F; and antibody light chain variable region gene VLThe germ line gene of (a) is derived from Musmus IGKV6-15 × 01F, and the connecting amino acids of V (D) J are CVRSGRYDKTYW and CHQYNRYPITF respectively. Obtained VHAnd VLThe gene can encode the correct mouse antibody variable region. Based on the cloned light and heavy chain variable region genes of the anti-human liver cancer monoclonal antibody IL6RA-C5Ab, the light and heavy chain variable region genes can be adoptedThe genetic engineering method is used for constructing and expressing various small molecular genetic engineering antibodies, such as single-chain antibodies, chimeric antibodies, Fab antibodies and the like, so as to be used for diagnosing and treating inflammatory diseases.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:
FIG. 1 ELISA screening of supernatants of hybridoma cell fusion strains; wherein, the samples No.2, No.5, No.9, No.14, No.15, No.18 and No.19 are positive clones for detection, wherein No.5 is the IL-6Ra-C5 hybridoma; the sample Positive is immune mouse serum diluted by 1: 10000; negative is an irrelevant murine IgG control;
FIG. 2 is agarose gel electrophoresis of RT-PCR amplified anti-human tumor monoclonal antibody IL6RA-C5Ab light and heavy chain variable region gene; wherein M is DL-2000Marker, IL6RA-C5Ab in lane 1 and IL6RA-C5Ab in lane 2;
FIG. 3 is the sequencing map of the variable region gene of the light chain of anti-human tumor monoclonal antibody IL6RA-C5 Ab;
FIG. 4 is the sequencing map of the anti-human tumor monoclonal antibody IL6RA-C5Ab heavy chain variable region gene;
FIG. 5 shows the FACs detection of anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab and hepatoma cell line HepG 2;
FIG. 6 is a Western blot detection of monoclonal antibody IL6RA-C5Ab in humanized IL-6Ra transgenic mice; wherein IL6ra-e (hIL6R) 1: a human IL-6Ra transgenic mouse; IL-6 RWT: c57 wild mouse; spleen, Liver and Kidney correspond to protein expression of Spleen, Liver and Kidney of three tissues of two mice detected by the antibody respectively;
FIG. 7 measurement of cytokine expression in groups following SIRS mice treated with mAb IL6RA-C5 Ab; LPS in the figure is 10mg/kg lipopolysaccharide; toilizumab is 2 mg/tuzumab. Wherein Panel A is the expression level of the cytokine TNF- α; panel B shows the expression level of cytokine IL-6; FIG. C is the expression level of the cytokine IL-1 β; panel D shows the expression level of the cytokine IFN-. gamma..
Detailed Description
Since 1975
And Milstein's technology for the creation of B-lymphocyte hybridomas, various monoclonal antibodies (MAbs) have emerged in succession, and they have played a positive role in the fundamental research and clinical applications of disease diagnosis and treatment. Monoclonal antibodies are distinguished from conventional serum antibodies, which are (polyclonal) hybrids of a number of different B lymphocytes directed against different epitopes of an antigen, whereas monoclonal antibodies are directed against a determinant of an antigen, produced by a B lymphocyte, and are therefore homogeneous, highly specific antibodies. Sensitized B lymphocytes secrete specific antibodies and each B lymphocyte produces antibodies only to a single epitope, but these antibody-secreting cells have a short lifespan and cannot grow for long periods of time in vitro culture conditions. With the development of genetic engineering antibody technology, phage antibody libraries, B cell sorting technology and the like have been developed greatly, but at present, hybridomas based on cell fusion are still one of the most feasible strategies for preparing therapeutic monoclonal antibodies.
Targeting the IL-6/IL-6R axis can serve as an effective target for intervention in a number of inflammatory or neoplastic diseases. The high affinity IL-6R receptor is composed of a ligand binding chain alpha chain (CD126) and a signal transduction chain gp130(CD130), both of which are members of the type I cytokine receptor family, and has wide expression. The alpha chain can be detached from the membrane surface or form soluble IL-6R (sIL-6R) due to different splicing of RNA, and can be combined with IL-6 to form a complex, and the biological effect of IL-6 is enhanced by combining with membrane type gp 130. Since IL-6 is a pleiotropic cytokine that regulates immune and inflammatory responses, it exerts biological effects by forming the IL-6Ra/IL-6/gp130 complex. It is thus also inspired us to develop new strategies for the mediated targeted treatment of tumors or inflammatory diseases with the IL-6/IL-6R system.
IL-6R can be divided into two types, namely a transmembrane type receptor and a soluble type receptor, wherein the soluble type receptor is similar to an enzyme cutting product of the transmembrane type receptor or a product of IL-6RmRNA splicing change. The cytoplasmic domain of IL-6R is very short and does not contain any kinase or specific fragment, so that it needs to be combined with gpl30, a receptor subunit responsible for signal transduction, to complete intracellular signal transduction and exert a series of biological effects of IL-6 factor, whether it is a transmembrane type receptor or a soluble type receptor. The IL-6R complex composed of the transmembrane receptor and gpl30 is called IL-6 classical receptor, and the signal transduction pathway mediated by the binding of IL-6 and the classical receptor is called classical signal pathway; while IL-6 in plasma binds to soluble receptors and further binds gp130 on the cell membrane, the signal transduction pathway mediated by it is called the trans-signaling pathway. Classical receptors are expressed primarily on the surface of neutrophils, macrophages, partial lymphocytes, and hepatocytes. Soluble receptors can be transported in plasma, and since gp130 is expressed on almost all cell surfaces, the biological range of action of IL-6 is expanded, e.g., synovial and endothelial cells do not express transmembrane IL-6R, but they express gpl30, so the IL-6R/IL-6 axis can still exert its effect.
The IL-6/IL-6Ra/gp130 complex can activate a series of downstream signaling pathways, including primarily the Ras-activated mitogen-activated protein kinase (MAPK) and Janus kinase (JAK) signaling and activator of transcription (STAT) signaling pathways. Ras/MAPK can phosphorylate and activate nuclear factor-IL 6(nuclear factor-IL6, NF-IL6), and activated NF-IL6 binds to IL-6 response elements of certain gene promoter regions to promote acute-phase protein synthesis. On the other hand, IL-6/IL-6Ra/gp130 activates JAK family tyrosine kinases, downstream transcription factors STAT1 and STAT3 and phosphatidylinositol 3-kinase (PI 3K), activated STATs migrate into the human nucleus, regulating transcription of a series of genes, and activated PI3K in turn activates the serine/threonine protein kinase B/AKT. These two signaling pathways mediated by IL-6 may mediate a wide range of immunological effects. As such, the IL-6/IL-6Ra/gp130 signaling pathway represents a potential target for therapeutic or prophylactic intervention in inflammation or cancer.
The expression of IL-6R is regulated by a variety of factors. Studies have shown that cellular IL-6R mRNA and protein expression is increased using glucocorticoid treatment, which may be blocked by glucocorticoid receptor-specific blockers. 17 beta-estradiol and dihydrotestosterone reduced IL-6R expression, thus resulting in an increase in IL-6R mRNA in ovariectomy in mice. Consistent with this, 17 β -estradiol down-regulates ciliary IL-6R levels in the oviduct of humans and mice. IL-6R expression in hepatocytes may be induced by IL-1 β or IL-6. The upregulation of IL-6R in human bronchial epithelial cells is mediated by IL-1 and IL-6. In NK92 cells, IL-6 treatment reduced IL-6R mRNA levels in IL-2 dependent natural killer cell lines. Chronic hepatitis b virus infection, the expression of IL-6R by CD4+ T cells is also enhanced. Infection with M.tuberculosis results in a decrease in Th17 responses in patients, which is associated with decreased IL-6R expression by CD4+ T cells. It has also been shown that IL-6R and gp130 are regulated in CD4+ T cells in peripheral blood, synovial fluid and synovial tissue of RA patients, and IL-6R expression is lower in CD4+ T cells in synovial tissue and fluid. Cell expression of gp130 in synovial fluid is down-regulated, and these cells cannot be activated by the classical and trans signals of IL-6. Because of the lack of expression of ADAM17, CD8+ T cells did not produce sIL-6R, while IL-6R expression on CD8+ T cells appears to be suppressed in a STAT 5-dependent manner. In relapsing remitting multiple sclerosis, it was found that enhanced IL-6R/STAT3 signaling and enhanced IL-6R expression had an effect on pathogenic effector T cell resistance. In addition, IL-2 has been shown to have a negative regulatory effect on the expression of T cell IL-6R mRNA and protein.
Furthermore, IL-2-/-T cells from mice show higher IL-6R expression. Since IL-2 also induces down-regulation of gp130 expression, its ability to inhibit T cell differentiation into Th17 cells appears to be based on changes in cytokine receptor expression. LPS has also been found to induce IL-6R expression in cultured pia mater cells. The expression of IL-6R is up-regulated by miR-124 or hepatocyte nuclear factor-4 alpha (HNF-4 alpha), and then the expression of IL-6R is influenced by the miR-124, miR-24 and miR-629. IL-6R was identified as a direct target for miR-125b, whose expression is often found to be down-regulated in hepatocellular carcinoma tissues. IL-6R is also a direct target of miR-34a, and a feedback loop of IL-6R/STAT3/miR-34 is found to promote invasion and metastasis of colorectal cancer. In endometrial cancer cellsThe inhibition of IL-6R by miR-124 is also described in cells. Elevated levels of E2F3 in prostate tumors help predict the clinical efficacy of patients. During prostate tumorigenesis, IL-6 signaling may be dependent on the role of E2F 3. In addition, the expression of IL-6R in breast cancer cells is upregulated, rendering the cells susceptible to the classical signaling pathway of IL-6, and thus, functional in the process of cellular senescence. Studies have shown that this phenotype is dependent on the activity of mammalian target of rapamycin (mTOR), and therefore we can demonstrate that activation of mTOR by Epidermal Growth Factor (EGF) is sufficient to induce expression of IL-6R.
Dysregulation of the IL-6R/IL-6 axis is associated with the development of various autoimmune diseases, and can play an important role in the development, development and metastasis of tumors in various ways such as regulating tumor microenvironment. The transmembrane IL-6R-mediated classical IL-6 pathway can promote anti-inflammatory or regenerative processes such as STAT 3-dependent intestinal epithelial cell regeneration, as well as activate hepatic acute phase responses and inhibit epithelial apoptosis. IL-6 secreted by cells acts as a pro-inflammatory action of IL-6 through a sIL-6R-mediated trans-signaling pathway, including recruitment of monocytes, inhibition of T-cell apoptosis, and inhibition of regulatory (Treg) differentiation. The cytokine IL-6 with high expression level is found in various tumors such as lung cancer, prostatic cancer, ovarian cancer and the like, and can cause the abnormality of a large number of normal cell activities, thereby promoting the progression of the tumors; in addition, IL-6 plays an important role in cardiovascular diseases such as vascular endothelial activation, endothelial dysfunction and hypertension-related thrombosis, which also means the possibility of the research of IL-6R/IL-6 axis targeted drugs, and the treatment targeting the IL-6R/IL-6 axis provides possible approaches and strategies for the diagnosis and treatment of tumors or inflammatory diseases.
In recent years, various biologics targeting the IL-6R/IL-6 signaling axis have been used for experimental treatment of autoimmune diseases including Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), inflammatory bowel disease, Giant Cell Arteritis (GCA), and the like. Fortunately, Toslizumab (TCZ) against recombinant humanized IgGl of IL-6R has been marketed in japan in 2008 and is now approved for the human chinese market at 4 months. Tocilizumab is a recombinant human-derived anti-human monoclonal antibody directed against immunoglobulin G1k subtype of soluble IL-6 receptor (sIL-6R) and membrane-bound IL-6 receptor (mIL-6R), and has been approved by the FDA for the treatment of RA and proven to be effective for other inflammatory diseases such as Castleman's disease.
In 2017, the FDA announces that tollgate monoclonal antibodies targeting human IL-6R are used for treating Cytokine Release Syndrome (CRS) caused by chimeric antigen receptor T (CAR-T) cell therapy, which is another important indication after the monoclonal antibodies are successfully used for autoimmune diseases such as rheumatoid arthritis and juvenile idiopathic arthritis, and then development of IL-6/IL-6R and its targeted inhibitors for the pathway is paid attention again.
The invention obtains a plurality of specific antihuman IL-6Ra monoclonal antibodies by combining the immunization method with a hybridoma technology, the monoclonal antibody with good specificity with a hepatoma cell strain HepG2 is obtained by screening through an ELISA method and is IL6RA-C5Ab, the monoclonal antibody can be combined with a target molecule IL-6Ra in vivo and in vitro, the antibody is positive to cell combination detected by immunofluorescence and a flow cytometer, and the mouse hybridoma cell strain IL-6Ra-C5 is delivered to China center for type culture Collection of Wuhan university (the preservation number is CCTCC NO: C2019126, the preservation date is 6 months and 12 days in 2019), and the cell activity is 100%.
First, ELISA screening of anti-IL-6 Ra antibody with high specificity
The cell strain is prepared by adopting a traditional cell fusion method in the fourth department of cell engineering research of military medical university, adopting limiting dilution clone cells, multiply diluting hybridoma cells, inoculating the hybridoma cells on a porous cell culture plate to ensure that each hole only contains one or more hybridoma cells, and finally selecting the hybridoma cell strain secreting a preset specific antibody for amplification culture by enzyme-linked immunosorbent assay (ELISA). The specific ELISA procedure was: the corresponding antigen solution (protein content is 1-10 mg/ml) is taken to carry out the overnight coating of a 96-well plate (coating buffer solution is 0.05mol/L, pH9.6 carbonate buffer solution), then 500 microliter Tween-20 is added with 1000ml of 0.01mol/L, pH7.6 phosphate buffer solution to wash the 96-well plate, 0.02mol/L containing 1% BSA, pH7.2 phosphate buffer solution is used for carrying out the room temperature blocking of the 96-well plate for 2h, hybridoma culture supernatant diluted by an equal time with a serum-free culture medium is added, and blank, negative and positive control wells are simultaneously arranged. Incubating for 1h at room temperature; washing with 0.02 mol/L0.05% Tween-20, pH7.2 phosphate buffer for 3 × 5min, adding horse radish peroxidase labeled goat anti-mouse IgG reagent, and placing in a wet box at 37 deg.C for 1 hr. Excess enzyme-labeled anti-antibody was spun off, washed 5 times with a washing solution, and then washed 2 times with distilled water. Adding 100 mul of newly prepared substrate TMB into each hole, placing the holes in a dark place at room temperature for acting for 5-30 minutes, adding 100 mul of 2mol/L sulfuric acid solution when the positive holes turn blue, stopping the reaction, and measuring the OD450 light absorption value by using an enzyme-labeled spectrophotometer.
ELISA screening showed that 8 positive cell lines were obtained after conventional cell fusion, and all of these cell lines showed good binding activity to IL-6Ra (as shown in FIG. 1). After three times of cloning, the clone positive rates of clone No.2 and clone No.5 reach 100%, namely hybridoma cell strains IL-6Ra-C2 and IL-6Ra-C5, and in the subsequent further identification, because monoclonal antibody IL6RA-C5Ab secreted by hybridoma cell strain IL-6Ra-C5 has better binding activity with cell strains HepG2 and U251MG, IL6RA-C5Ab is selected for further biological function verification experiments. A conventional antibody subtype identification kit is selected, and the antibody subtype of IL6RA-C2Ab is IgG2b, and the antibody subtype of IL6RA-C5Ab is IgG1 Kappa.
Cloning of anti-human tumor monoclonal antibody IL6RA-C5Ab light and heavy chain variable region gene
Taking IL-6Ra-C5 hybridoma cells (5X 10) in logarithmic growth phase6) Total RNA is extracted by adopting a guanidine isothiocyanate one-step method, and a small amount of total RNA is taken to be subjected to ultraviolet spectrophotometer quantification and 1 formaldehyde modified agarose gel electrophoresis detection. Followed by oligo (dT)15(Promega corporation) was a random primer, and first strand cDNA was synthesized by reverse transcription. Then, a set of designed universal primers are used for successfully amplifying the light chain variable region gene and the heavy chain variable region gene (V) of the liver cancer monoclonal antibody IL6RA-C5AbH，VL). Amplification product VH(about 410bp) and VL(about 400bp) was electrophoresed through a low melting point agarose gel (1.5%), and the target fragment was separated by gel cutting. By passingGel purification kit (Promega corporation) was recovered and purified, and then identified by gel electrophoresis (see FIG. 2). The target fragment was then cloned into the T-vector and analyzed by sequencing (see FIGS. 3 and 4).
Oligo(dT)15The reverse transcription protocol for the random primers is as follows: mu.g of total RNA (2. mu.L) and 0.5. mu.g of random primer oligo (dT) were added to a 20. mu.L reaction system in sequence15(1μL),4μLMgCl 22 μ L (25mM) of 5 XDNTPs, 2 μ L of 10 XTPS, 0.5 μ L of RNase inhibitor, reverse transcriptase AMV 15U (0.75 μ L), water-supplemented to 20 μ L, mixing, water bath at 42 deg.C for 1h, boiling for 3min, and standing the reaction product at-20 deg.C for use.
The PCR amplification reaction is carried out according to a conventional method: using the above-mentioned product as template, respectively using 3 pairs of heavy chain primers and 5 pairs of light chain primers to amplify antibody light chain and heavy chain variable region genes. The designed PCR amplification primer sequence of the variable region gene of the IL6RA-C5Ab monoclonal antibody is as follows:
mouse heavy chain V region 5' primer:
(1)5'-GGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT-3'；
(2)5'-GGGGATATCCACCATG(AG)ACTTCGGG(TC)TGAGCT(TG)GGTTTT-3'；
(3)5'-GGGGATATCCACCATGGCTGTCTTGGGGCTGCTCTTCT-3'；
mouse heavy chain V region 3' end primer:
5’GAC(ACT)CATGGGG(CG)TGT(TC)GTGCTAGCTG(AC)(AG)GAGAC(AGT)GTGA-3’；
mouse light chain V region 5' primer:
(1)5'-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3'；
(2)5'-GGGGATATCCACCATGGATTTTCAAGTGCAGATTTTCAG-3'；
(3)5'-GGGGATATCCACCATGGAG(AT)CACA(GT)(AT)CTCGGGTCTTT(GA)TA-3'；
(4)5'-GGATATCCACCATG(GT)CCCC(AT)(AG)CTCAG(CT)TC(CT)CT(TG)GT-3'；
(5)5'-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3'。
mouse light chain V region 3' primer:
5'-GGATACAGTTGGTGGTGCAGTCGACTTACGTTT(GT)GTTTCA(AG)CTT-3'；
wherein: underlined are restriction enzyme sites.
The reaction system is as follows: mu.L of template cDNA, 2.5. mu.L of dNTPs (0.4 mM each), 5. mu.L of 10 XBuffer, 1.25U of Ex Taq DNA polymerase, 5. mu.L (about 30pmol) of 5 'end and 3' end primers, 50. mu.L of water, mixing, subjecting to flash centrifugation, adding 1-2 drops of paraffin wax, and placing on a PCR apparatus for reaction. The reaction conditions are 94 ℃ for 1min, 54 ℃ for 1min, 72 ℃ for 1min, 35 cycles, and finally 72 ℃ extension for 10 min.
The sequencing scheme of the target fragment T vector clone is as follows: the PCR product was recovered after gel electrophoresis separation and ligated into the pMD18-T vector. The connection reaction system is as follows: 1 mu L of pMD18-T vector, 3 mu L of PCR product gel purified heavy chain (or light chain), 1 mu L of deionized water, 5 mu L of connecting buffer solution, mixing uniformly, standing overnight at 4 ℃, transforming Escherichia coli JM109, screening recombinant clones, and then adopting a universal sequencing primer to perform sequencing analysis.
Through antibody subtype identification, the corresponding molecular subtype of the antibody is mouse IgG1Kappa, and corresponding germline genes are respectively from Musmus IGHV3-2 x 02F and Musmus IGKV6-15 x 01F.
II, identifying cells and tissues of anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab by Western-blot
Western-blot was performed according to the conventional method [ SambrookJ, Fritsch E F, Maniatis T.molecular Cloning: A Laboratory Manual.2nd ed, New York: Cold Spring Harbor Laboratory Press,1989]Then, tumor cell line HepG2 or fixed point knock-in human IL-6Ra transgenic mice (southern model animal center: IL6Ra-e (hIL6R)1) were subjected to conventional Western-blot identification after RIPA tissue lysis. Wherein, for mouse tissue, 5mm is usually taken3The left and right tissues were ground thoroughly in liquid nitrogen, 200. mu.l of tissue lysate was added, shaking thoroughly, lysed on ice for 30min, centrifuged at 12000rpm/min for 20min, and the supernatant was measured for protein concentration using bicinchoninic acid (BCA) method kit. Then, 20. mu.g of the sample was subjected to 10% SDS-PAGE using reduced or non-reduced SDS. After electrophoresis, the gel is transferred to a nitrocellulose membrane, the transfer membrane is cut into equal parts according to the width of the corresponding staining gel, and after being sealed for 1 hour by 5 percent skim milk, monoclonal antibody IL6 diluted by 1:1000 is usedRA-C5Ab was treated at room temperature for 1 hour. The treated membrane was washed three times with TBST buffer and incubated with HRP-labeled goat anti-mouse IgG (H + L) for 1 hour at room temperature. 4X 10 min/time washing of the membrane. Finally, the blot was detected using an enhanced chemiluminescent substrate (ECL kit, Millipore). The control primary antibody used for immunoblotting was directed against IL-6R α (mouse monoclonal antibody, Santa Cruz Biotechnology, sc-373708, diluted 1: 500), β -actin (rabbit monoclonal, abcam, ab179467, diluted 1: 3000), and the secondary antibody was an HRP-conjugated antibody (goat anti-rabbit IgG, horse anti-mouse IgG, #7074, Cell Signaling Technology, all diluted 1: 2000).
The result shows that the expression of the transgenic mouse protein is detected by Western Blot, compared with a wild C57 mouse, the anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab prepared by the item can detect the expression of human IL-6Ra in the main tissues of the transgenic mouse (figure 6), which not only indicates that the mouse already contains the expression of human IL-6Ra, but also indicates that the prepared antibody can be specifically bound to the molecule.
Thirdly, identifying tumor cell FACs of anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab
Collecting 2X 106Cells to be tested (e.g., HepG2 cells, etc.) were washed once with PBS and centrifuged at 800rpm for 5min (or 200 × g). Fixing the cells with 100 μ l of 4% paraformaldehyde at room temperature for 20min, centrifuging at 800rpm for 5min, discarding the supernatant, resuspending the cells with PBS, centrifuging at 800rpm for 5min, and repeating for 1 time; then, the cells are sealed by sheep serum for 15min, centrifuged at 800rpm for 5min, and the supernatant is discarded. Add purified 100u g/ml anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab antibody 30 u l (hybridoma secretion supernatant 1:2 dilution), ice placed for 30min,800rpm centrifugation for 5min, use PBS heavy suspension cells, 800rpm centrifugation for 5min, wash away the unbound antibody. After adding 100ul of a goat anti-mouse secondary antibody labeled with a fluorescent label (FITC) (1:100) and leaving it on ice for 30min in the dark, 800 rpm: centrifuge for 5min, remove supernatant, wash twice with PBS, discard supernatant. 300uL of PBS was added for cell resuspension; and (3) detecting on a computer, wherein mouse multiple antiserum of an immunized mouse is selected as a positive control, and mouse IgG is selected as a negative control.
The result shows that the anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab prepared by the method can detect the antigen expression on a hepatoma cell line HepG2 and a glioma cell line U251, and the prepared antibody has good target specificity.
Fourth, anti-human IL-6Ra monoclonal antibody IL6RA-C5Ab for mice systemic inflammatory response syndrome SIRS and evaluation
Humanized IL-6Ra transgenic C57 mice (IL6Ra-e (hIL6R)1) were randomly divided into a normal control group, three different doses of IL-6Ra mAb treatment groups (concentrations), and tolzumab (positive control group): A. a normal control group; B. LPS (10 mg/kg); C. LPS (10mg/kg) +0.1 mg/IL-6 Ra; D. LPS (10mg/kg) +0.5 mg/L-6 Ra; E. LPS (10mg/kg) +2.5 mg/IL-6 Ra; F. LPS (10mg/kg) +2mg tuzumab/mouse; each group had 5 mice, which were injected intraperitoneally with anti-human IL-6Ra monoclonal antibodies for 8h and 12h, and then sera were collected and tested for the expression of the mouse serum inflammatory cytokines TNF- α, IL-1 β, INF- γ, and IL-6 (Dakewe Bio-engineering Co, Ltd.).
The serum ELISA detection result shows that compared with a control group, the SIRS symptom induced by LPS can be obviously relieved after the anti-human IL-6Ra monoclonal antibody is injected. The levels of the proinflammatory cytokines TNF- α, IL-1 β, IL-6 and IFN- γ were relatively high at 8 and 12h after LPS injection. IL-6Ra mAb treatment significantly reduced the serum concentrations of these four cytokines in LPS-injected mice (FIG. 7).
In conclusion, the protein medicine reconstructed from the light chain variable region gene and the heavy chain variable region gene into a certain form can be directly used for diagnosis and targeted therapy of tumors and inflammations related to IL-6Ra targets. In addition, the polypeptide and the derivative thereof encoded by the light chain and heavy chain variable region genes are used as a carrier, and cytotoxic drugs, toxins, radionuclides, enzymes, biological response regulators and the like are crosslinked to be used as guiding drugs, so that the polypeptide and the derivative thereof can be used for diagnosing and treating diseases related to IL-6 Ra.
Starting from the polypeptide encoded by the light and heavy chain variable region genes, the novel antibody which can be recombined mainly has the following forms:
(1) a chimeric antibody. The V region of mouse MAb is connected with the C region of human Ig to form human-mouse chimeric antibody. The specificity and affinity of the mouse MAb are completely reserved, and adverse reactions such as HAMA are reduced, so that the composition has a good effect in treating diseases such as tumors.
(2) A humanized antibody. The humanized modification aiming at the gene structure of the variable region comprises CDR transplantation, surface amino acid residue inlaying, framework region exchange, positioning reservation, epitope guide selection and the like, thereby not only reducing the murine origin of the variable region, but also keeping the specificity and the affinity of the murine MAb.
(3) A small molecule antibody. Mainly composed of VH-CH1 and VL-C1 constituting a Fab antibody, conjugated with a polypeptide (GLy4Ser)3Joint connection VHGenes and VLSingle-chain antibody derived from gene, VHAnd VLFv fragment antibody formed by non-covalent bonding of VHOr VLA single domain antibody consisting of one functional domain, a minimal recognition unit consisting of a single CDR, and the like.
(4) A multivalent minibody. Mainly diabodies, (scFv)2Flex minibody, LD minibody, F (ab')2、F(ab')3、(scFv)4And the like. Because of the multivalent antigen binding site, high affinity and moderate molecular size, the polypeptide has the characteristics of penetrating tumor tissues and slow clearance in the kidney, thereby having higher clinical application value.
(5) Bispecific antibodies. Is an antibody with dual specificity and dual functions, also called bifunctional antibody.
(6) Recombinant antibody fusion proteins. A recombinant protein which is formed by linking a gene fragment such as Fab or Fv to a gene of another protein such as a toxin or an enzyme which is not an antibody and has a specific biological activity directed to a target site.
(7) A recombinant immunotoxin. The gene of coded antibody and toxin is recombined to produce the product, which features high effect, low non-specific toxicity, high stability in body, easy penetration to tumor and safe use in body.
(8) A phage antibody. The V region gene of Ig is connected with gene III or gene VIII on filamentous phage DNA, and after the host bacterium is transfected, the fusion protein product of Fab or scFv is expressed on the coat protein of the membrane surface. The desired specific antibody is screened out by affinity adsorption of the product to multiple rounds of related antigens.
The immunoconjugates or targeted drugs formed by cross-linking various anti-inflammatory or tumor effector substances with the above-mentioned polypeptide recombinant or derived antibody molecules encoded by the light and heavy chain variable region genes as carriers mainly have the following forms:
(1) antibody-nuclide conjugates. The conjugate can effectively guide nuclide to the local part of tumor tissue, thereby reducing normal tissue damage and related adverse reactions caused by external radiation of radiotherapy, and can carry out positioning diagnosis and guide treatment on tumor. This method is known as radioimmunography and radioimmunotherapy. Common nucleic acids conjugated with monoclonal antibodies are131I，111In，90Y，188Re and186re, and the like.
(2) Antibody-chemotherapeutic drug conjugates. The conjugate can specifically guide to tumor, reduce damage to normal tissue, and reduce toxic and side effects of chemotherapy drugs. Chemotherapeutic drugs commonly coupled are: phosphoric acid amides in alkylating agents; against methotrexate and 5-fluorouracil in metabolism; antibiotics adriamycin, epirubicin, daunorubicin; vincristine and mitomycin.
(3) An antibody-toxin conjugate. The conjugate is also called immunotoxin. It has strong cell killing effect and is independent of biological auxiliary mechanism. The tumor killing mechanism is different from that of radiotherapy and chemotherapy, so that the tumor with poor radiotherapy and chemotherapy effect can be used and has wide application prospect. The commonly used toxins include ricin, diphtheria toxin, abrin, saporin, pseudomonas exotoxin, streptolysin, perforin, etc.
(4) antibody-Biological Response Modifier (BRM) conjugates. BRM has better curative effect on independently regulating the immunity of organisms and killing tumor cells. However, only part of the tumor reaches the target position of the tumor after the tumor is delivered into the body, so the killing effect of the tumor cannot be fully exerted and the tumor has toxic and side effects. The BRM is coupled with the monoclonal antibody, and the monoclonal antibody carries the BRM to reach a target site to play a role in killing tumors, so that the factors have stronger and more specific effects. The commonly used BRM includes INF and IL-2.
(5) The antibody targets the enzymatically hydrolyzed prodrug. Specificity of antibody to drugThe conjugate of sexual activation enzyme is injected into body, and the prodrug is injected after a certain time interval, so that the prodrug is converted into high-concentration anticancer active drug at the tumor site to kill tumor cells. Currently, glutamine derivatives of predrug such as mechlorethamine benzoate, etoposide phosphate, mitomycin phosphate, daunorubicin, doxorubicin, 5-fluorocytosine, and cephalosporine. The activating enzyme is carboxypeptidase G2Alkaline phosphatase, penicillin amidase, beta-lactamase, cytosine deaminase, beta-glucosidase, aminopeptidase and the like.
(6) Immunoliposomes. The MAb is coupled to the surface of the liposome, so that the guidance of the MAb and antigen specificity combination and the characteristic that a large amount of medicaments can be wrapped by the liposome are fully integrated, and the relevant medicaments are embedded, so that the targeting property and the curative effect of the antibody medicament can be further improved.
The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Nucleotide sequence list electronic file
<110> the fourth military medical university of the Chinese people liberation army
<120> anti-human IL-6Ra monoclonal antibody and application thereof
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Ser Asp Phe Ala Trp Asn
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<213> mice
<220> CDR2 of the heavy chain variable region
<400>
Tyr Ile Ser Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu Lys Ser
<210>3
<211>8
<212>PRT
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<220> CDR3 of the heavy chain variable region
<400>
Ser Gly Arg Tyr Asp Lys Thr Tyr
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<212>PRT
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<220> CDR1 of the light chain variable region
<400>
Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
<210>5
<211>7
<212>PRT
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<220> CDR2 of the light chain variable region
<400>
Ser Ala Ser Tyr Arg Tyr Ser
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<220> CDR3 of the light chain variable region
<400>
His Gln Tyr Asn Arg Tyr Pro Ile Thr
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<220> heavy chain variable region Gene sequence
<400>
atg aga gtg ctg att ctt ttg tgg ctg ttc aca gcc ttt cct ggt atc 48
ctg tct gat gtg cag ctt cag gag tcg gga cct ggc ctg gtg aaa cct 96
tct cag tct ctg tcc ctc acc tgc act gtc act gac ttc tca atc acc 114
agt gat ttt gcc tgg aac tgg atc cgg cag ttt cca gga aag aaa ctg 192
gag tgg atg ggc tac ata agt tac agt ggt agc act acc tac aac cca 240
tct ctc aaa agt cga atc tct atc tct cga gac aca tcc atg aac cag 288
ttc ttc ctg cag ttg aat tct gtg act act gag gac aca gcc aca tat 336
tat tgt gtt aga tcg gga agg tac gac aag act tac tgg ggc caa ggg 384
act ctg gtc act gtc tct gca 405
<210>8
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<220> light chain variable region gene sequence
<400>
atg ggc atc aag atg gag tca cag act cag gtc ttt gta tac atg ttg 48
ctg tgg ttg tct ggt gtt gat gga gac att gtg atg acc cag tct caa 96
aaa ttc atg tcc aca tca gta gga gac agg gtc agc gtc acc tgc aag 144
gcc agt cag aat gtg ggt act aat gta gcc tgg tat caa cag aaa cca 192
ggg caa tct cct aaa gca ctg att tac tcg gca tcc tac cgg tac agt 240
gga gtc cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc act 288
ctc acc atc acc aat gtg cag tct gaa gac ttg gca gac tat ttc tgt 336
cac caa tat aac agg tat cct atc acg ttc gga ggg ggg acc aag ctg 384
gaa ata aaa 393
<210>9
<211>135
<212>PRT
<213> mice
<220> polypeptide encoded by heavy chain variable region gene sequence
<400>
Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile Leu
1 5 10 15
Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
20 25 30
Ser Leu Ser Leu Thr Cys Thr Val Thr Asp Phe Ser Ile Thr Ser Asp Phe
35 40 45 50
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp Met Gly
55 60 65
Tyr Ile Ser Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu Lys Ser Arg
70 75 80 85
Ile Ser Ile Ser Arg Asp Thr Ser Met Asn Gln Phe Phe Leu Gln Leu Asn
90 95 100
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Val Arg Ser Gly Arg
105 110 115
Tyr Asp Lys Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
120 125 130 135
<210>10
<211>131
<212>PRT
<213> mice
<220> polypeptide encoded by light chain variable region gene sequence
<400>
Met Gly Ile Lys Met Glu Ser Gln Thr Gln Val Phe Val Tyr Met Leu Leu
1 5 10 15
Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe
20 25 30
Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln
35 40 45 50
Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
55 60 65
Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg
70 75 80 85
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val
90 95 100
Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys His Gln Tyr Asn Arg Tyr Pro
105 110 115
Ile Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
120 125 130 131
Claims (8)
1. An anti-human IL-6Ra monoclonal antibody comprising:
a heavy chain variable region, wherein the 3 complementarity determining regions of the heavy chain variable region are: CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in SEQ ID NO: 3 is shown in the specification;
a light chain variable region, wherein the 3 complementarity determining regions of the light chain variable region are: CDR1 is as set forth in SEQ ID NO: 4, CDR2 is as shown in SEQ ID NO: 5, CDR3 is set forth in SEQ ID NO: and 6.
2. An anti-human IL-6Ra monoclonal antibody comprising a heavy chain variable region encoded by a heavy chain variable region gene and a light chain variable region encoded by a light chain variable region gene:
the gene sequence of the heavy chain variable region gene is shown as SEQ ID NO: 7 is shown in the specification;
the gene sequence of the light chain variable region gene is shown as SEQ ID NO: shown in fig. 8.
3. An anti-human IL-6Ra monoclonal antibody comprising: as shown in SEQ ID NO: 9 and the heavy chain variable region as set forth in SEQ ID NO: 10, or a light chain variable region.
4. A hybridoma cell expressing an anti-human IL-6Ra monoclonal antibody according to any one of claims 1 to 3, wherein the hybridoma cell has a accession number of CCTCC NO: C2019126.
5. use of the monoclonal antibody against human IL-6Ra according to any one of claims 1 to 3 for the preparation of a medicament for the treatment, diagnosis or prevention of Systemic Inflammatory Response Syndrome (SIRS), liver cancer and/or glioma.
6. An antibody-nuclide conjugate, an antibody-chemotherapeutic drug conjugate, an antibody-toxin conjugate, an antibody-Biological Response Modifier (BRM) conjugate, an antibody-targeting enzyme, or an immunoliposome comprising the anti-human IL-6Ra monoclonal antibody of any of claims 1-3.
7. A kit, comprising:
a) the anti-human IL-6Ra monoclonal antibody of any one of claims 1-3, and b) a conjugate comprising an antibody linked to a signal producing compound, wherein the antibody in the conjugate is different from the anti-human IL-6Ra monoclonal antibody of any one of claims 1-3.
8. A kit, comprising: a) the anti-human IL-6Ra monoclonal antibody of any one of claims 1-3, and b) a conjugate comprising an antigen linked to a signal producing compound.
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| CN104119438A (en) * | 2009-01-29 | 2014-10-29 | 米迪缪尼有限公司 | Human anti-iL-6 antibodies with extended in vivo half-life and their use in treatment of oncology, autoimmune diseases and inflammatory diseases |
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| CN104119438A (en) * | 2009-01-29 | 2014-10-29 | 米迪缪尼有限公司 | Human anti-iL-6 antibodies with extended in vivo half-life and their use in treatment of oncology, autoimmune diseases and inflammatory diseases |
| WO2011006273A1 (en) * | 2009-07-15 | 2011-01-20 | Eva Kovacs-Benke | Combination of a human interleukin-6 antagonist and a human interleukin-6 receptor antagonist in therapy of tumour diseases |
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