WO2015042807A1 - 抗cd20的全人源单克隆抗体及其应用 - Google Patents

抗cd20的全人源单克隆抗体及其应用 Download PDF

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WO2015042807A1
WO2015042807A1 PCT/CN2013/084203 CN2013084203W WO2015042807A1 WO 2015042807 A1 WO2015042807 A1 WO 2015042807A1 CN 2013084203 W CN2013084203 W CN 2013084203W WO 2015042807 A1 WO2015042807 A1 WO 2015042807A1
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seq
variable region
chain variable
antibody
amino acid
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PCT/CN2013/084203
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French (fr)
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冯晓
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北京安保康生物医药科技有限公司
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Priority to CN201380072095.1A priority patent/CN105143269B/zh
Publication of WO2015042807A1 publication Critical patent/WO2015042807A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the technical field of antibody drugs. Specifically, it relates to a group of new human monoclonal antibody molecules which are useful for the diagnosis and treatment of lymphoma. Background technique
  • CD20 is a non-glycosylated phosphoprotein with a molecular weight of 33 ⁇ 37 kDa. It has four transmembrane regions. The amino terminus and the carboxy terminus are located on the inner side of the plasma membrane, between the third transmembrane region and the fourth transmembrane region. , has a loop region consisting of 43 amino acid residues, which constitutes its main epitope.
  • As a surface differentiation antigen of B lymphocytes it is initially expressed in the pre-B cell stage, and ends when the B cell terminal differentiates into plasma cells, and has been considered to be a unique marker on the surface of the B cell line. It is mainly involved in regulating the proliferation and differentiation of B lymphocytes and plays an important role in the immune system.
  • non-Hodgkin's lymphoma are B-cell sources, and about 95% of these cells have surface CD20 expression [1 ' 2] .
  • the expression of CD20 is different depending on the lymphoma cells.
  • the surface expression of follicular lymphoma cells is high.
  • the surface expression of small cell lymphoma cells is low, and it is not expressed in stem cells and plasma cells.
  • Chronic B lymphoblastic leukemia cells The expression is much lower than that of normal B cells and other B lymphoma cells.
  • the expression level of CD20 determines the extent of antibody and complement killer tumor cells to some extent [3] . Monocytes, resting, and activated T cells, naked cells, and non-lymphocytes do not express CD20 molecules.
  • CD20 is an immunotherapy B cell lymphoma.
  • the ideal site of action, especially for the treatment of inert, relapsed and refractory B cell lymphoma has a positive effect.
  • Anti-CD20 treatment can eliminate malignant B cells and some normal B cells, but since stem cells and B cell precursors do not express CD20, they do not cause long-term B cell loss.
  • the main indications for clinical application are: 1. Treatment of follicular B-cell non-Hodgkin's lymphoma with anti-CD20 monoclonal antibody alone; 2. Anti-CD20 monoclonal antibody combined with chemotherapy for diffuse large B-cell lymphoma And chronic B lymphocytic leukemia (B-CLL).
  • IDEC-C2B8 also known as Rituximab, is marketed as rituximab, and was approved by the FDA in 1997. It is the first monoclonal antibody approved by the US FDA for the treatment of tumors [4] . It is a human-chimeric chimeric antibody comprising the variable region of the murine anti-CD20 monoclonal antibody 2B8 (Ibritumomab) and the constant region of the human IgGl heavy chain and kappa chain for the treatment of sputum cell lymphoma.
  • Rituximab exerts significant anti-tumor efficacy in vivo by inhibiting cell proliferation or triggering multiple cell destruction mechanisms including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and apoptosis [5] . Recent clinical studies have confirmed that the drug has a wide spectrum of treatments, Cellular diseases can achieve better results.
  • the drug is produced by IDEC Pharmaceuticals, Inc. under the generic name Ibritumomab Tiuxetan.
  • This product is used in tumor therapy by the mouse IgGl- ⁇ monoclonal antibody 2B8 (Ibritumomab) linked to the isotope 9Q Y. Its monoclonal antibody moiety has a high specific affinity for CD20 [6] .
  • Bexxar Tositumomab and 131 1 Tositumomab
  • Bexxar for the treatment of CD20-positive follicular NHL with or without metastasis of cancer cells, resistance to Rituximab, and relapse after chemotherapy. It is formed by covalent coupling of a murine monoclonal antibody primary antibody B1 monoclonal antibody CTositumomab, lgGla- ⁇ ) with a radioisotope 131 1 .
  • Rituximab can be used alone, the recommended dose is 375 mg / m 2 per week, intravenous injection, a total of 4 times, treatment of relapse or drug-resistant low-grade malignant CD20 positive B cell NHL; Phase II clinical trials show that overall efficiency For 48%, 6% are fully effective, 42% are partially effective, and the remission period is usually 1 year.
  • the single-agent effective rate is about 50% ⁇ 70%, and maintenance treatment can further improve the curative effect.
  • the side effects are milder, and can be reduced as the amount of antibody input is reduced. There are no or few infection complications during treatment. Therefore, Rituximab has become an irreplaceable role in the treatment of NHL as a first-line drug.
  • Xu Zhiqiao et al [8] used a prospective study of concurrent control, 22 patients with B-cell NHL were divided into study group (Rituximab group) and control group, and study group 11 patients with CHOP regimen (cyclophosphamide, more flexible) Bixing, Changchun Neobase and prednisone were treated with Rituximab; 11 patients in the control group were treated with CHOP alone.
  • CHOP regimen cyclophosphamide, more flexible Bixing, Changchun Neobase and prednisone
  • the complete remission rate (CR) of the Rituximab group was 72.7% (8/11), and the total effective rate was 90.9% (10/11).
  • the control group was 36.4% (4/11), and the total effective rate was 54.6% (6/). 11), the difference in efficacy between the two groups was statistically significant.
  • Preliminary results suggest that Rituximab combined with CHOP regimen is effective in the treatment of CD20-positive B-cell NHL, and
  • Rheumatoid arthritis is a systemic autoimmune disease characterized by synovitis as a major pathological change characterized by chronic destructive joint disease.
  • B cells are the main source of autoantibodies such as rheumatoid factor (RF) and anti-citrullin antibody (anti-CCP).
  • RF rheumatoid factor
  • anti-CCP anti-citrullin antibody
  • Edwards et al [1()] reported a randomized, double-blind, controlled study of 161 large samples of the oral methotrexate group ( ⁇ 10 mg/week), the ituximab group (lg dl, dl5), and the Rituximab combination ring.
  • the results showed that the Rituximab treatment group reached 65% to 76% of ACR20 after 24 weeks, and 38% of the methotrexate treatment group alone.
  • Rituximab correlates with B cell clearance, demonstrating the therapeutic role of Rituximab in RA and the important role of B cells in the pathogenesis of A.
  • a series of studies on the therapeutic effects of Rituximab on RA have also shown that Rituximab is a stable treatment for RA and is a new approach to biotherapy.
  • SLE Systemic lupus erythematosus
  • B cells play a key role in the pathogenesis of SLE: secretion of a large number of pathogenic autoantibodies and various cytokines such as interleukin-10 (IL-10), interleukin-6 (IL-6), tumor necrosis factor (TNF- ⁇ ) Highly selective antigen presentation, presenting antigen to T cells, while stimulating regulation of T cell and dendritic cell activation [11] .
  • IL-10 interleukin-10
  • IL-6 interleukin-6
  • TNF- ⁇ tumor necrosis factor
  • SLAM systemic lupus erythematosus activity score
  • Immunologic thrombocytopenic purpura is an autoimmune hemorrhagic disease.
  • T lymphocyte regulation disorder in ITP patients leads to dysfunction of B lymphocytes, producing IgG antibodies against autologous platelets, which in turn increases platelet destruction.
  • Rituximab can quickly and permanently remove B lymphocytes from circulation, reduce the production of autoantibodies, and thus reduce the destruction of platelets.
  • Taube et al [13] treated 22 children with chronic ITP using Rituximab.
  • CD20 positive B lymphocytes in peripheral blood decreased significantly after 1 to 2 weeks after Rituximab use, and gradually returned to normal after 2 to 3 months.
  • the monoclonal antibody (MAb) thus prepared is an antibody against a single antigenic determinant, which has high specificity and homogeneity, and can be prepared in large quantities, which has become an antibody research field.
  • MAb monoclonal antibody
  • OKT3 the first monoclonal antibody drug OKT3 prepared by this technology was approved by the US FDA and used to treat immune rejection after organ transplantation.
  • the antibody since the antibody is mouse-derived, it causes a serious immune reaction in the patient, which greatly hinders the development and utilization of the antibody drug.
  • the application of monoclonal antibody humanization technology has broken the bottleneck of antibody drug industrialization. So far, according to the degree of humanization, it can be roughly divided into three technologies: chimeric, humanized and all-human, and the proportion of human sequences in the genes is 75%, 95% and 100%, respectively [22] .
  • the chimeric antibody is obtained by ligating the variable region of the murine hybridoma monoclonal antibody gene and the human constant region, and then expressing it in mammalian cells; whereas the humanized antibody is in addition to changing the constant region of the antibody.
  • the FR region of the variable region is further converted to the adult source [2()] , thereby reducing immunogenicity; while the human-derived antibody preparation has four pathways, one is collected from A Fab or ScFv library is constructed in a patient's blood sample and the antibody library is screened by phage display to generate a human MAb [18] .
  • the first all-human source, MAb, Humira was developed through phage display.
  • the second strategy for the production of human antibodies is to immunize with transgenic mice containing human immunoglobulin gene loci, which can produce an immune response of human antibodies, so that human MAb can be produced by conventional hybridoma technology [17] ], the transgenic mice derived from humanized MAb in clinical trials.
  • the third way is to isolate and culture human peripheral blood lymphocytes or tonsil lymphocytes, then use the antigen to immunize, generate an immune response, and then fuse with human myeloma cells to produce hybridoma cells, which are screened to obtain human monoclonal.
  • the antibody is immediately subjected to molecular cloning of the antibody gene, and other mammalian cells stably expressing for a long period of time are used to express the antibody of interest, which is completely produced in a human cell environment.
  • the fourth type of pathway is to perform antigen-specific screening of culture supernatants by separating and low-density (about 500 B cells per 96 well) human peripheral blood lymphocytes or tonsil lymphocytes, and molecular cloning of positive pore B cells.
  • the antibody gene is then used to express mammalian cells that are stably expressed for a long period of time to express the antibody of interest.
  • the fully human monoclonal antibody of the present invention was obtained based on the above third route and subjected to random mutation screening.
  • Monoclonal antibodies produced using human-human hybridomas are considered to be fully human antibodies that are superior to chimeric and humanized antibodies in both amino acid sequence and glycosylation profile.
  • it is difficult to find fusion partner cells that stably pass human-human hybridomas and due to problems such as low fusion efficiency, difficulty in cell culture, and low productivity, human-human hybridoma cells stably stabilizing and capable of continuously producing monoclonal antibodies are prepared.
  • the system has always been difficult, and there are very few successful examples.
  • the present invention overcomes the above difficulties by rapidly performing screening of human-human hybridomas expressing human antibodies, including specificity and function, and molecularly cloning the selected hybridoma cells to preserve the antibody genes.
  • the three antibodies against CD20 that have been approved for marketing are currently created by Biogen-Idec.
  • Zevalin and Bexxar are murine monoclonal antibodies, and mainly through the antibody-mediated radioisotope to the tumor part, play a radioactive cytotoxicity to directly kill tumor cells. Due to factors such as radiotoxicity and the production of human anti-mouse antibodies, the clinical efficacy is poor and the indications are narrow.
  • Rituxan ie Rituximab
  • the present invention is aimed at the creation of new molecules for the inadequacy of Rituximab: (1) the murine human chimeric antibody is a fully human antibody (2) by the promotion of new drugs, making it directed against CDC, ADCC and other tumor cells. The ability to apoptate is enhanced to improve the efficacy of tumoricidal. Summary of the invention
  • the present invention provides a group of novel antibodies against CD20, which are fully human antibodies, and as a new drug, the multiple CD20-specific cytotoxic effects of these antibodies are stronger than those of the American drug, Miro, and are the same as or similar to rituximab. Better medicine. It can be used for the treatment and diagnosis of inflammatory diseases, autoimmune diseases, cell proliferative disorders, cardiovascular diseases, blood diseases, especially non-Hodgkin's lymphoma, B cell lymphoma, rheumatic and rheumatoid diseases, systemic erythema Diseases such as lupus, immune thrombocytopenic purpura and/or multiple sclerosis.
  • the heavy chain constant region of the antibody of the present invention is the constant region of the human IgG1 heavy chain
  • the light chain constant region is the constant region of the human kappa chain
  • the amino acid sequence of the heavy chain variable region is Seq ID NO: 1, Seq ID NO: 2 Seq ID NO: 3 Seq ID NO: 4 and one of Seq ID NO: 9
  • the amino acid sequence of the light chain variable region is Seq ID NO: 5, Seq ID NO: 6, Seq ID NO: 7, Seq ID NO: 8 And one of the Seq ID NO: 10.
  • preferred combinations are 1) and 2), and more preferably 1).
  • the antibodies of the present invention further comprise a CDR sequence comprising a heavy chain variable region comprising Seq ID NO: 1-4 and 9, and a light chain variable region comprising Seq ID NO: those antibodies of the CDR sequences of 5-8 and 10.
  • CDR sequences are detailed in Seq ID NO: 11-26.
  • the antibody of the present invention is not limited to human IgG and human kappa chain, and the region other than the variable region of the antibody of the present invention may be other types such as human IgM or the like.
  • the nucleotide sequence encoding the same can be designed based on the amino acid sequence of the above antibody, and the nucleotide sequence is optimized for different expression hosts.
  • the antibodies of the invention may be covalently linked to isotopes, immunotoxins and/or chemical agents, may also be coupled to solid media, semi-solid media, and may also function as antibodies of the invention. Sex fragments, which are known in the art.
  • the present invention provides a method of using this method to make a fully human antibody and as a new drug.
  • the most important technical difficulties overcome by this method are (1) in vitro effective immunization of human lymphocytes; (2) human hybridoma genes are unstable, and screening must be carried out including specificity and function, and screening will be performed.
  • the hybridoma cells are subjected to molecular cloning to preserve the antibody gene; (3) the human antibody having been screened for certain pharmacological effects is further optimized by molecular biological antibody engineering technology.
  • the preparation of the antibody and the related detection method are as follows: by extracting peripheral blood lymphocytes of different humans or human tonsil lymphocytes, mixing them in vitro, and co-cultivating the antigen and the molecule having the adjuvant function to produce antibodies against the antigen. By fusion with myeloma cells, a temporarily stable human hybridoma is produced, and the culture supernatant contains the secreted human antibody.
  • CD20 positive hybridoma cell growth wells were screened by cell ELISA (double screening of CD20 positive cells and CD20 negative cells), and the best 10 were screened by relative antigen affinity, cytotoxicity test (ADCC, CDC and apoptosis assay).
  • the cells were grown in wells and immediately subcloned to screen for monoclonal cell lines that maintained antigen specificity.
  • the sequencing and molecular cloning of the antibody gene is immediately performed into an appropriate expression vector, and the plasmid DNA is amplified and purified and stored in -8CTC for permanent storage. Cytotoxicity based on relative antigen affinity Can be tested (ADCC, CDC and apoptosis experiments) to comprehensively evaluate the antibody genes of the best three monoclonal cell lines, and use this as a template to perform in vitro antibody affinity maturation engineering.
  • ADCC relative antigen affinity
  • CDC apoptosis experiments
  • the libraries were screened for antigen specificity and relative affinity ranking.
  • the heavy chain variable region ( VH ) and the light chain variable region (VL) of each of the best 5 ⁇ 10 antibodies are fully aligned to form a full length antibody.
  • a small amount of purified antibody was prepared using transient, culture and micro-affinity purification (Protein A) and screened by cytotoxicity test (ADCC, CDC and apoptosis assay). After screening a group of antibody molecules with good cytotoxicity, and searching for the variable regions of these antibodies, especially the CDR regions, it is determined that the antibody with CD20-specific cytotoxicity is the first new molecule. . There are one or more new antibody molecules in this group of new molecules. (1) Multiple CD20-specific cytotoxic effects are stronger than merocide; (2) Subcutaneous xenografts and survival test results show that they have the same characteristics as the US The drug, rituximab, has the same and better efficacy.
  • the antibody After determining the amino acid sequence of an antibody of the present invention, the antibody can be prepared by artificially synthesizing a polynucleotide encoding the antibody and selecting a suitable host for efficient expression, as is well known in the art.
  • Figure 1 to Figure 3 show the results of CDC test of Raji cells;
  • Figure 1 shows the CDC functional screening of different human hybridoma supernatants;
  • Figure 2 shows the purification of human antibodies H2L3, H2L6, H2L10, H8L3 by CHO cells after affinity maturation.
  • Figure 3 is a CDC functional screening of purified human antibodies H8L15, H11L6, H11L10, H19L3 and H19L15 expressed by CHO cells after affinity maturation;
  • Figure 4 to Figure 6 are the results of ADCC test of Raji cells;
  • Figure 4 is a functional screening of ADCC for different human hybridoma supernatants;
  • Figure 5 is a representation of purified human antibodies H2L3, H2L6, H2L10 for CHO cells after affinity maturation.
  • Figure 6 is an ADCC functional screen for expression of purified human antibodies H8L15, H11L6, H11L10, H19L3 and H19L15 in CHO cells after affinity maturation;
  • Figure 7 Heavy chain derived from cell line 1.105.3 The mutations of H1 ⁇ H20 are sorted according to affinity;
  • FIG screened by heavy chain and light chain gene mutation mutant gene were combined according to the ranking of affinity; corresponding result data and the K D values of Table 7;
  • Figure 10 shows the results of pharmacodynamic tests of the mouse Raji cell invasive tumor model of antibody H2L10 in the treatment of human B cell lymphoma, which is an observation of the body weight and health status of the mouse.
  • Intravenous injection of 10 7 Raji lymphoma cells forms a mouse lymphoma disease model.
  • the five groups were 1. saline group, 2. rituximab group, 3.H2L10 (ie, representative antibody of the present invention) low dose group, 4.H2L10 medium dose group and 5.H2L10 high dose group.
  • Dosing was started 7 days after intravenous injection of cancer cells. Intraperitoneal injection once a week. The figure shows the changes in body weight of each group of animals.
  • Figure 11 shows the results of the survival test of the mouse Raji cell invasive tumor model of antibody H2L10 in the treatment of human B cell lymphoma.
  • Intravenous injection of 10 7 Raji lymphoma cells forms a mouse invasive lymphoma disease model.
  • the five groups were 1. saline group, 2. rituximab group, 3.H2L10 low dose group, 4.H2L10 medium dose group and 5.H2L10 high dose group. Dosing was started 7 days after intravenous injection of cancer cells. Intraperitoneal injection once a week. Animals in the saline group died from the 23rd day and died on the 34th day.
  • Figure 12 shows the results of an anti-tumor experiment of orthotopic transplantation of Raji cells in human B cell lymphoma mice treated with antibody H2L10. After the mice were inoculated, the tumor growth was observed. When the tumor volume was about 40-100 mm 3 , the tumor volume was selected according to the tumor volume. The tumor volume was too large and the tumor-free ones were not selected. After screening, they were randomly divided into 3 groups: 1. saline group, 2. rituximab group, 3.H2L10. The dose was 5 mg/kg, and it was intraperitoneally injected once a week. The results clearly show that both rituximab and H2L10 can significantly inhibit the growth of subcutaneous xenografts of Raji cells. The efficacy of H2L10 is similar to that of rituximab.
  • Figures 13A and 13B show ADCC test results of Raji cells against antibodies A137, H2L10 and rituximab.
  • Figure 14 shows the results of CDC testing of Raji cells by antibodies A137 and rituximab.
  • Figure 15 shows the results of the affinity tests for A137 and rituximab.
  • the table shows the relevant parameters Bmax and K D and confidence intervals.
  • Figure 16 shows the results of orthotopic transplantation of A137 and rituximab in the treatment of human B cell lymphoma mouse raji cells.
  • Each mouse was intravenously injected with 10 7 Raji lymphoma cells to form a mouse invasive lymphoma disease model, and administered intraperitoneally 5 days later.
  • Experiments were performed in 3 groups of mice (8 per group): A. saline control group; B. rituximab group (dose 1 mg/kg body weight); C. A137 group (dose 1 mg/kg body weight).
  • Figure 17 is a graph showing changes in body weight of mice treated with A137 and rituximab in the treatment of human B cell lymphoma mouse raji cells orthotopically transplanted tumors.
  • Each mouse was intravenously injected with 10 7 Raji lymphoma cells to form a mouse invasive lymphoma disease model, and administered intraperitoneally 5 days later.
  • Experiments were performed in 4 groups of mice (8 per group): A. saline control group; B. rituximab high dose group (3 mg/kg body weight); C. rituximab low dose group (1 mg/kg body weight); 0. 137 groups (1 mg/kg body weight).
  • the human CD20 (Novusbio) antigen was purchased, diluted with PBS, and filtered through a 0.22 ⁇ needle sterile filter. 1.1.2 Preparation of 293F cells expressing CD20 membrane protein
  • CD20 forward arch I substance 5 '-TCAGGAGTTTTGAGAGCAAAATG-3 '
  • the cDNA of CD20 was used as a template for amplification, purification, and cloning into the pCT.l vector (Invitrogen, K300001). After sequencing, the plasmid was prepared in large quantities, and then transfected into FreeStyleTM 293-F cells according to the instructions (Invitrogen, 790-07) After 48 hours, 1 mg/ml of G418 (Invitrogen, 10131035) was added for 2 weeks, and stable G418-resistant clones were screened and frozen.
  • the CD20 membrane protein was extracted from CD20-293F cells according to the kit instructions using the Cell Membrane Protein and Cytoplasmic Protein Extraction Kit (purchased from Biyuntian Institute of Biology). Filter with a 0.22 ⁇ m filter and store in a 1.5 ml cell cryotube at -80 °C.
  • lymphocyte separation solution GE Lymphocytes were isolated and taken from 30 ⁇ 10 6 ; fresh peripheral blood cells were aseptically extracted, and lymphocytes were separated by lymphocyte separation (GE), and 30 ⁇ 10 6 were taken.
  • the lymphocyte medium is formulated as: 1 liter of DMEM containing 20% fetal bovine serum (FBS), 1% of 200 millimoles of L-glutamic acid (Sigma, cat. # G 2150), 1% of 100x non-essential amino acids ( Sigma, cat. # M 7145), 1% lOOx penicillin streptomycin mixture (Sigma, cat. # P 7539), (10,000 U/ml penicillin + 10 mg/ml streptomycin) > 10 units/ml IL-6 (Boehringer Mannheim, cat. # 1299972), 20 units/ml of human recombinant interleukin 2 (R&D system, cat.
  • 16 individual lymphocytes from two sources in 1.2 were mixed and mixed cultured in RPMI-1640 medium containing 20% FBS, and the final concentration of lymphocytes was 5 ⁇ 10 5 /ml, and the following step 2.1 was obtained.
  • Mouse peritoneal spheroid cells final concentration of lxlO 5 / ml
  • non-essential amino acids final concentration of lug / ml
  • CD20 antigen protein immunization: final concentration of lug / ml
  • cell membrane immune group from 10
  • Six cell membrane proteins/ml of CD20-293 cells were co-cultured for 6-12 days in a carbon dioxide incubator (new culture medium was changed on days 5-6).
  • BALB/c mice purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd.
  • They were immersed in 75% alcohol for 5 minutes.
  • scissors were used for aseptic operation. Open the abdomen skin, expose the peritoneum, inject 10 ml of HAT medium (20% FBS, HAT lx, the rest is DMEM) into the abdominal cavity with a syringe, repeatedly suck, aspirate the medium into a 50 ml centrifuge tube, trypan blue cell count, adjust The cell concentration was 2 x 10 5 /ml, added to a 96-well plate, 100 ul / well, and placed in a carbon dioxide incubator overnight. This procedure is applicable to all experiments that require the use of mouse peritoneal sputum cells.
  • Myeloma cells P3X63Ag8.653 were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, and cultured to logarithmic growth phase, centrifuged at 1000 rpm for 5 minutes, suspended in DMEM without FBS, 1000 rpm. Centrifuge for 5 minutes, flick the bottom, suspend in DMEM medium without FBS, trypan blue count, and the cell viability is 95%.
  • Lymphocytes were collected 6 days after co-culture with CD20 antigen, trypan blue counting live cells, centrifugation at OO rpm for 5 minutes, discarding the supernatant, flicking the bottom, and suspending in a small amount of DMEM medium without FBS; with the same number of myeloma
  • the cells were well mixed, supplemented with DMEM medium to 40 ml, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. 2 ml of sterile pronase solution (CalBiochem, 53702) was added, and the cell pellet was slightly suspended for 2 minutes, and 3-5 ml of FBS was added to terminate.
  • Electrofusion solution cell count, adjust cells to 2x10 6 /ml, divided into 2ml / tube; set according to the number of seconds of 2ml volume, start electrofusion device (BTX); after fusion, centrifuge at lOOOOrpm for 5 minutes, discard the supernatant ; after adding a small amount of HAT medium before suspending the cells, the appropriate volume of HAT medium was added according to the amount of added fusion when lymphocytes, lxlO 6 cells / plate, was added to support cell servo paved 96, placed in a carbon dioxide incubator for cultivation.
  • BTX start electrofusion device
  • Coating 12 days after fusion, specific antibodies were detected by double-ELISA, one set was coated with 293 cell membrane expressing CD20, one set was coated with untransfected 293 cell membrane; overnight coated at 4 °C, concentration was 50 Ten thousand cell membranes / 50 ul / well, the coating solution was 1% BSA PBS with a pH of 8.3. Blocking: Plates were washed 3 times with PBST, blocked with 10% skim milk powder PBS, incubated for 1 hour at room temperature; then plated 3 times with PBST.
  • the CD20-293 coated plate was positive and the 293 coated plate negative well was antigen specific.
  • the positive wells were selected and expanded to 4 96 wells. After 2 days, the reamed wells were tested to confirm the positive wells and expanded to 24 wells. When the 24-well cells grow to about 1/3 of the bottom of the well, an ELISA is performed. This method is also applicable to the detection of late antibodies.
  • step 2.5 The cell culture supernatant of step 2.5 which was still positive after re-ELISA identification was used, and the human Ig quantitative detection ELISA kit (Cat. No. E88-104) of Bethyl Laboratories was used, and the specific steps were carried out according to the kit instructions.
  • human Ig quantitative detection ELISA kit Cat. No. E88-104 of Bethyl Laboratories was used, and the specific steps were carried out according to the kit instructions.
  • Sample preparation The antibody to be compared, ie the purified antibody of known concentration or the cell culture supernatant of the known antibody concentration after quantitative detection in step 2.6, is diluted to 4-8 identical concentrations (120 nM, 40 nM, 13.1 nM, 4.4nM). Then perform the ELISA test as described in 2.4. Since all samples were previously adjusted to the same antibody concentration, the difference in OD values will be caused by the affinity of the antibody. The higher the OD value, the higher the relative affinity. The Prism software was used to map and calculate the affinity for each antibody.
  • 10 cell growth wells having the highest antigen affinity and functional efficacy were selected, i.e., 1.1, 1.4, 1.6, 1.36, 1.72, 1.89, 1.105, 1.134, 1.146, 1.176.
  • Mouse peritoneal spheroid cells (using HT medium) were collected 1 day before the subcloning or on the same day, and the cell concentration was lx lO 5 /ml, and added to a 96-well plate, lOOul/well. After 1.1, 1.4, 1.6, 1.36, 1.72, 1.89, 1.105, 1.134, 1.146, 1.176 positive hybridoma cell lines screened by the step 2.7 were respectively subjected to trypan blue staining, the cells were plated to the plate at the following concentrations. Medium: density 5 / hole, 1 block; 1 / hole, 1 block; 0.5 / hole, 2 blocks.
  • the cells were observed from the bottom of the well through an inverted microscope, and the pores were confirmed to be monoclonal with a marker. Only the cell culture supernatants of these monoclonal wells were transferred, 50 ⁇ l/well, and subjected to ELISA (see Method 2.4). From the cell-state and OD values, the subcloned hybridoma cell lines (1.1, 1.4, 1.6, 1.36, 1.72, 1.89, 1.105, 1.134, 1.146, 1.176) were selected based on the cell status and OD value. The supernatant of the best 3-5 monoclonal cell wells was identified by the next step (2.9).
  • a monoclonal cell line identified as IgGl- ⁇ was selected, which were: 1.1.81, 1.4 .68, 1.6.14, 1.36.2, 1.72.108, 1.89.45, 1.105.3, 1.134.42, 1.146.78, 1.176.109.
  • the 10 monoclonal cell lines were expanded to 24 wells (using DMEM medium containing 20% FBS), and the cells were grown well, and after detection, expanded to square flasks.
  • cryopreservation solution 10% DMSO, 60% FBS, 30% DMEM, pre-cooled on ice, the cells were suspended, placed in a centrifuge tube, trypan blue count, centrifuged, discarded, and pre-frozen Liquid, press 2 ⁇ 5xl0 6 tubes/tubes for cryopreservation.
  • the cells were placed in a programmed cooling box (Nalgene) and placed in a -70 ° C refrigerator overnight, and transferred to liquid nitrogen the next day.
  • the monoclonal cell strains that maintain antigen specificity are screened out for 1.1.181, 1.4.68, 1.6.14, 1.36.2, 1.72.108, 1.89.45, 1.105.3, 1.134.42, 1.146.78, 1.176.109. Sequencing and molecular cloning are performed immediately, the antibody gene is introduced into an appropriate expression vector, and the plasmid DNA is amplified and purified and stored at -80 ° C for permanent storage.
  • the supernatant of the cells cultured in a 225 cm 2 square flask for about 10 days was taken out, centrifuged, and the cell debris was removed, and the antibody was purified by rProtein A affinity chromatography.
  • the collected antibody solution was filtered through a 0.22 um filter and stored aseptically.
  • Ramos aji and Daudi cells were purchased from ATCC with 10% FBS, 1% sodium pyruvate and 1%
  • HEPES buffered DMEM HEPES buffered DMEM.
  • the CellTiterGlo kit was purchased from Promega (Madison, WI). Normal human serum is isolated from whole blood of healthy blood donors.
  • Ramos, Raji and Daudi cells were plated into 96-well plates at 1 x lOO/well.
  • the cells were incubated with different concentrations of the antibody to be tested for 10 minutes in a 37 ° C, 5% C0 2 cell culture incubator, in which the antibody to be tested was The cell supernatant antibody obtained in Example 2 or the affinity matured antibody obtained in Example 6, the positive control was rituximab, and the negative control was purified human IgG (prepared from serum of healthy humans). Normal human serum was then added to give a final concentration of 10% in the culture.
  • Cell lysis rates were determined by CellTiterGlo kit after incubating cells, different concentrations of different antibodies and human serum for 60 minutes in a 37 ° C, 5% C0 2 cell culture incubator. If the supernatant of the hybridoma culture is screened, it is necessary to adjust the concentration of the plurality of supernatant antibodies to be screened to 1 ( ⁇ g/ml according to the result of the antibody concentration in the supernatant of the quantitative ELISA, and then compare the antibodies at the same concentration. the CDC effect to filter out high CDC antibody efficacy antibody hybridoma strain. calculated and plotted using Prism software EC 50. the results shown in Figure 1, Figure 2, Figure 3 and table 1, table 2.
  • Table 1 CDC functional screening of supernatants from different human hybridoma cell lines
  • Table 2 CDC functional screening of purified human antibodies expressed by CHO cells after affinity maturation (see Table 6 for the source of sorted antibodies in the table)
  • Target cell preparation Raji cells were suspended in serum-free DMEM medium to a concentration of 1 ⁇ 10 5 /ml, Calcein AM (Sigma) was added to a final concentration of 10 UM, 37 ° C, and incubated for 50 minutes. The supernatant was centrifuged, and the cells were suspended in serum-free DMEM medium to 104/75 ul and plated in a black clear bottom 96-well plate (Corning) at 75 ul/well. The test antibody and the positive control were diluted in PBS to different concentrations, and 25 ul/well was added to the plate and incubated at room temperature for 30 minutes.
  • Effector cell preparation Aseptically take fresh normal human venous blood, anticoagulate blood with anticoagulant, add osettesep Human NK cell Enrichment cocktail (50 ml), add an equal volume of whole blood containing 2% FBS in PBS, add Lymphocyte separation solution, centrifuge at 2150 rpm for 30 minutes; transfer the middle layer cells to a conical tube, add PBS containing 2% FBS, centrifuge at 2150 rpm for 10 minutes, discard the supernatant; suspend the cells with a small amount of PBS, use trypan blue Count, adjust the cell concentration to 10 5 /75ul,
  • Table 4 ADCC functional screening examples of purified human antibodies expressed by CHO cells after affinity maturation (see Table 6 for the source of sorted antibodies in the table)
  • the cells were suspended in 200 ul PBS, loaded into a cell counting plate, and the apoptotic rate was determined by counting the total number of cells and the number of apoptotic cells stained by fluorescence microscopy.
  • the results are shown in Table 5. The results showed that: H2L10 was about 2 times more potent than rituximab in apoptosis and CDC experiments, and was equivalent to rituximab in the ADCC test.
  • Table 5 Comparison of apoptosis, complement-mediated cytotoxicity, and antibody-mediated cytotoxicity of H2L10 and rituximab (H2L10 source see Example 6).
  • Apoptosis, antibody-mediated cytotoxicity, and complement-mediated cytotoxicity assays are the three major anticancer drug pharmacodynamic tests.
  • the half effective drug concentration (EC 5 ) is a standard measure comparing the pharmacodynamics of different drugs.
  • the experiments shown in the table are the results of back-to-back pharmacodynamic experiments performed on H2L10 with rituximab as a control.
  • anti-CD20 monoclonal antibody cell line 1.105.3 was selected as a template for antibody affinity maturation experiments based on the results of affinity and cell function experiments. Sequence analysis of heavy and light chains of antibody genes.
  • RNA of anti-CD20 monoclonal antibody cells 1.105.3 was extracted using Invitrogen's Trizol® reagent kit (15596-026); then random primers from Takara's 5 'RACE FULL kit (D315) were used to total RNA as a template, reverse transcription into the first strand cDNA, and according to the instructions of the kit, add a linker at the 5'end; then use the adaptor primers provided in the kit to specifically bind to the human heavy chain gene IgG1 constant region. Primers and light chain gene kappa chain constant region-specific primers were amplified to obtain the heavy chain variable region and the light chain variable region of the anti-CD20 monoclonal antibody (referred to herein as the "wild type" antibody) gene. Heavy and light chain specific primers for PCR amplification are:
  • the PCR amplification product was cloned into a T vector (Takara, D101A), sequenced, and sequenced to obtain a consensus heavy chain variable region and a light chain variable region.
  • the constructed single-chain antibody scFv was used as a template, random mutation was performed, and the mutated single-chain antibody was subjected to phage display, and then a higher affinity antibody was screened by ELISA.
  • the heavy chain variable region and the light chain variable region specific primers were designed, and the Sfil site and the Xhol site were added to the heavy chain variable region 5, the terminal and the 3, respectively.
  • the variable region 5, the terminal and the 3, the end plus the Nhel site and the Notl site, the heavy chain variable region (VH) and the light chain variable region (VL) amplification primers are as follows:
  • VH Reverse Primer CCACCACTCGAGGC GCTCGAGACGGTGACCAGGGT
  • VL forward primer TGGCGGGTCGACG GATATTGTGATGACCCAGACT
  • VL reverse primer TGTTCTGCGGCCGC TTTGATCTCCACCTTGGTC
  • the fully variable VH and VL clones were used as templates, and the heavy chain variable region and the light chain variable region were amplified by PCR, and then cleavage and stepwise cloning into the phage display vector pFL249 (gene synthesis construct) for sequencing verification.
  • the correctly sequenced clone A0026/pFL249-scFv was transformed into TG1 competent cells (Stratagene, 200123), and the monoclonal was picked for culture, induction, and supernatant.
  • the Raji cell membrane was used as antigen for ELISA.
  • Statgene's GeneMorph II Random Mutagenesis Kit (200550) was used to perform error-prone PCR amplification with VH forward primer and VL reverse primer, and the amplified PCR product was purified, digested and ligated into pFL249 vector. Then, it was electrotransformed into TG1 competent cells, diluted to the appropriate concentration, plated, and cultured overnight at 37 °C.
  • the monoclonal antibody was picked from a monoclonal density plate to a 96-well bacterial culture plate for culture and induced expression, and the supernatant was collected for ELISA. In the OD value higher than the wild type, the OD value was high to low. 200 monoclonals were selected for sequencing. After analyzing the sequencing results, after removing the non-human clones of the FR region mutation, VH and VL of 20 mutant clones were selected to construct a full-length antibody according to the OD value from high to low.
  • the wild-type VH and VL clones, and the 20 VH and 20 VL clones after mutating were used as templates, and the heavy chain variable region and the light chain variable region were amplified by PCR, and then digested and cloned into The human IgG1 constant region and the human kappa constant region pCI vector (; Promega, E1731) were sequence verified.
  • the antibody concentration in 40 antibody supernatants was quantified according to the procedure of 2.6 in Example 1 above, and then the relative affinity ranking was carried out according to the method of step 2.7 in Example 1 above.
  • the results are shown in Fig. 7, Fig. 8 and Table 6:
  • the affinity of the supernatant of the 40 mutants was 10 to 20 times higher than that of the original cell line 1.105.3. Five of the highest affinity heavy chains H2, H8, H11, H16, H19 and five light chains L3, L6, L10, L15, L18 with the highest affinity were selected.
  • Table 6 Affinity sequences of heavy and light chains of the mutated (K D is the dissociation constant (unit: nM), Prism software by ELISA with an affinity sorting data calculated)
  • the heavy chain constant region of the first 10 antibodies shown in Table 7 is the constant light chain constant region of the human IgG1 heavy chain, which is the constant region of the human kappa chain, the amino acid of the heavy chain variable region and the light chain variable region.
  • the sequences are as follows: 6.5.1 Heavy chain variable region sequences and their complementarity determining regions (CDRs)
  • Seq ID NO: 1 is the amino acid sequence of the variable region of H19
  • Seq ID NO: 2 is the amino acid sequence of the variable region of H11
  • Seq ID NO: 3 is the amino acid sequence of the variable region of H8
  • Seq ID NO: 4 is the amino acid sequence of the variable region of H2
  • Seq ID NO: 9 is the amino acid sequence of the variable region of A137H
  • Seq ID NO: 11 is the CDR1 sequence of H19 H11 H8 and A137H
  • Seq ID NO: 12 is the CDR1 sequence of H2
  • Seq ID NO: 13 is the CDR2 sequence of H19
  • Seq ID NO: 14 is the CDR2 sequence of H2 H11 Seq ID NO: 15 is the CDR2 sequence of A137H
  • Seq ID NO: 16 is the CDR2 sequence of H8
  • Seq ID NO: 17 is the CDR3 sequence of H19, H2, H8 and A137H
  • Seq ID NO: 18 is the CDR3 sequence of HI 1
  • Seq ID NO: 5 is the amino acid sequence of the variable region of L3
  • Seq ID NO: 6 is the amino acid sequence of the variable region of L10
  • Seq ID NO: 7 is the amino acid sequence of the variable region of L6
  • Seq ID NO: 8 is the amino acid sequence of the variable region of L15
  • Seq ID NO: 10 is the amino acid sequence of the variable region of A137L
  • Seq ID NO: 19 is the CDR1 sequence of L3
  • Seq ID NO: 20 is the CDR1 sequence of L10
  • Seq ID NO: 21 is the CDR1 sequence of L6
  • Seq ID NO: 22 is the CDR1 sequence of L15
  • Seq ID NO: 23 is the CDR1 sequence of A137L
  • Seq ID NO: 24 is the CDR2 sequence of L3, L6, L10, L15 and A137L
  • Seq ID NO: 25 is the CDR3 sequence of L3, L6, L10 and A137L
  • Seq ID NO: 26 is the CDR3 sequence of L15.
  • Intravenous injection of 10 7 Raji lymphoma cells forms a mouse lymphoma disease model.
  • the five groups were 1. saline group (no antibody), 2. rituxa group (single dose was 3.5mg/kg mouse body weight), 3.H2L10 low dose group (single dose was 1mg/kg), 4 The H2L10 medium dose group (single dose was 3.5 mg/kg) and the 5.H2L10 high dose group (single dose was 10 mg/kg).
  • Dosing was started 7 days after intravenous injection of cancer cells. Intraperitoneal injection once a week. The results are shown in Figure 10.
  • Intravenous injection of 10 7 Raji lymphoma cells forms a mouse lymphoma disease model.
  • the five groups were 1. saline group (no antibody), 2. rituximab group (single dose was 3.5mg/kg), 3.H2L10 low dose group (single dose was lmg/kg), 4.H2L10 The dose group (single dose was 3.5 mg/kg) and the 5.H2L10 high dose group (single dose was 10 mg/kg).
  • Dosing was started 7 days after intravenous injection of cancer cells. Intraperitoneal injection once a week. The results are shown in Figure 11.
  • mice were inoculated with human B-cell lymphoma, the tumor growth was observed, and the tumor volume was about 40-100 mm 3 .
  • the tumor size was screened, and the tumor volume was too large and no tumorigenicity was selected.
  • they were randomly divided into 3 groups: 1. saline group, 2. rituximab group, 3.H2L10, and the doses of the rituximab group and the H2L10 group were 5 mg/kg mouse body weight. Intraperitoneal injection once a week. The experimental results are shown in Figure 12.
  • the efficacy test in animals was carried out using A137.
  • the human B-cell lymphoma model mice injected with normal saline began to die at about 21 days, and all died at 40 days; while the mice injected with rituximab began to die at about 44 days, and all died at about 130 days;
  • the group of mice injected with A137 began to die at approximately 47 days, and more than 20% of the mice survived after 140 days (see Figure 16). This proves that the efficacy of A137 is better than that of rituximab.
  • A137 is better than the 3-fold dose of rituximab in terms of mouse body weight maintenance: By 150 days, the body weight of the group receiving A137 is still slow, and the weight loss is maintained within 15%. The high-dose rituximab group lost more than 15% after about 100 days, and lost more than 35% at about 130 days.
  • the present invention is directed to the creation of new molecules for the inadequacy of Rituximab: (1)
  • the murine human chimeric antibody is a fully human antibody (2) by the promotion of new drugs, targeting CDC, ADCC and other tumor cells. The ability to apoptate is enhanced to improve the efficacy of tumoricidal.
  • Pharmacodynamic test for treating human B cell lymphoma Raji cell invasive tumor model and pharmacodynamic test for treating human B cell lymphoma Raji cell orthotopic transplantation tumor, both in survival and in inhibiting tumor growth Shows no less than or better efficacy than rituximab.
  • the antibody molecule of the present invention can be used as a drug candidate molecule to have a better therapeutic effect on lymphoma and autoimmune diseases, and is beneficial for the treatment of human diseases.

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Abstract

本发明提供了一种抗CD20的全人源单克隆抗体，其通过人杂交瘤技术制备。所述抗体具有优异的亲和力和特异性，对淋巴瘤细胞具有显著的细胞毒功效，可用于制备治疗或诊断非霍奇金氏淋巴瘤、B细胞淋巴瘤、风湿性和类风湿性疾病、系统性红斑狼疮、免疫性血小板减少性紫癜和多发性硬化症等疾病的药物组合物。

Description

抗 CD20的全人源单克隆抗体及其应用 技术领域
本发明涉及抗体药物的技术领域。 具体地说， 涉及一组全人源单克隆抗体新分子， 其可用于淋巴瘤的诊断和治疗。 背景技术
1. CD20
CD20是一种分子量为 33〜37 kDa的非糖基化磷蛋白，有 4个跨膜区，氨基端和 羧基端都位于细胞质膜内侧， 在第三跨膜区和第四跨膜区之间， 有一个由 43个氨基 酸残基组成的环区， 构成其主要的抗原表位。 作为 B淋巴细胞表面分化抗原， 它起 始表达于 pre-B细胞阶段，到 B细胞终端分化成浆细胞时结束，一直被认为是 B系细 胞表面特有的标识。 它主要参与调节 B淋巴细胞的增殖与分化， 在免疫系统起重要 作用。 80%~85%的非霍奇金氏淋巴瘤 (NHL)为 B细胞来源， 并且这些细胞的约 95% 均有表面 CD20表达 [1'2]。 CD20的表达因淋巴瘤细胞的不同而有所差异， 滤泡性淋巴 瘤细胞表面表达较高， 小细胞淋巴瘤白血病细胞表面表达较低， 在干细胞和浆细胞中 不表达，慢性 B淋巴白血病细胞中的表达远低于正常的 B细胞和其他 B淋巴瘤细胞， CD20 的表达高低在一定程度上决定了抗体和补体杀伤瘤细胞的程度 [3]。 单核细胞、 静息以及激活的 T细胞、裸细胞以及非淋巴细胞都不表达 CD20分子。 CD20与抗 CD20 抗体结合后内化现象不明显，细胞表面 CD20分子数量并不因为与抗体结合而大量减 少， CD20也不会发生明显细胞表面脱落的现象， 因此， CD20是免疫治疗 B细胞淋 巴瘤的理想作用位点， 特别是对治疗惰性、 复发难治性 B 细胞淋巴瘤有较肯定的疗 效。
2.治疗性抗 CD20抗体
抗 CD20治疗可清除恶性 B细胞和部分正常 B细胞， 但由于干细胞和 B细胞前 体不表达 CD20， 因而不会造成长期 B细胞损耗。 其临床应用主要适应证为： 1.单纯 应用抗 CD20单克隆抗体治疗滤泡性 B细胞型非霍奇金淋巴瘤； 2. 抗 CD20单克隆 抗体与化疗联合应用治疗弥漫大 B细胞型淋巴瘤及慢性 B淋巴细胞白血病 (B-CLL)。
2.1 Rituximab (美罗华）
IDEC-C2B8, 又名 Rituximab, 商品名为美罗华， 1997年由 FDA批准上市， 是第 一个被美国 FDA批准用于治疗肿瘤的单克隆抗体 [4]。 它是一个人鼠嵌合抗体， 包含 鼠源抗 CD20单克隆抗体 2B8 (Ibritumomab)的可变区和人源 IgGl重链及 κ链的恒定 区， 用于 Β细胞淋巴瘤的治疗。 Rituximab在体内通过抑制细胞增殖或触发多种细胞 破坏机制， 包括抗体依赖性细胞毒作用 (ADCC)和补体依赖性细胞毒作用 (CDC)以及 细胞凋亡发挥明显的抗肿瘤功效 [5]。 近年的临床研究证实， 该药治疗谱较广， 对 B淋 巴细胞性疾病均可取得较好的疗效。
2.2 ZEVALIN
2002年 2月， FDA批准了第一个放射性免疫治疗药物 Zevalin。 该药由 IDEC制 药公司生产， 通用名 Ibritumomab Tiuxetan。 FDA批准此药用于治疗复发性或难治性 低恶性度 /滤泡性或转化的 B细胞 NHL， 其中包括 Rituximab难治性的滤泡性 NHL。 该产品由小鼠 IgGl-κ单克隆抗体 2B8 (Ibritumomab)连接同位素 9QY用于肿瘤治疗。 其单抗部分对 CD20具有高的特异亲和性 [6]。
2.3 BEXXA
2003年 6月 27日， FDA批准 Bexxar (Tositumomab和 131 1 Tositumomab)用于治 疗癌细胞已经或未发生转移、 对 Rituximab有耐药性、 化疗后又复发的 CD20阳性滤 泡性 NHL。 它由鼠源单克隆抗体一抗 B1单克隆抗体 CTositumomab, lgGla-λ )与放射 性同位素 1311共价偶联而成。
2.4 A ZERA
2009年 10月 26日， FDA批准 ARZERA Ofatumumab)用于治疗慢性淋巴细胞性 白血病 （CLL)。 它由 KM人源转基因鼠免疫通过杂交瘤技术产生的人单克隆抗体。 和 Rituximab相似其在体内通过抑制细胞增殖或触发多种细胞破坏机制， 包括抗体依 赖性细胞毒作用 (ADCC)和补体依赖性细胞毒作用 (CDC)以及细胞凋亡发挥明显的抗 肿瘤功效。 但其在治疗非霍奇金氏淋巴瘤 (NHL)的三期临床试验失败。 目前其主要用 于对一线化疗药物耐受的慢性淋巴细胞性白血病。
3. Rituximab在临床的应用
3.1 单独应用治疗非霍奇金氏淋巴瘤 (NHL)
Rituximab可单独应用， 推荐的使用剂量是每周 375毫克 /平方米， 静脉内注射， 共 4次， 治疗复发或耐药性低度恶性 CD20阳性 B细胞 NHL; II期临床试验表明， 总体有效率为 48%， 其中 6%为完全有效， 42%为部分有效， 缓解期通常为 1年。 对 于初治的低度恶性 NHL， 单药有效率为 50%〜70%左右， 维持治疗能够进一步提高 疗效。 副作用较温和， 可随抗体输入量的减少而减轻， 治疗中没有或少有感染并发症 发生。 因而 Rituximab作为一线药物应用治疗 NHL已有着无法替代的作用。
2004年， 欧盟许可 Rituximab联合标准化疗以治疗侵袭性 NHL。 一些临床试验 用来评估 Rituximab单独或联合治疗惰性、 侵袭性 NHL和其他一些 B细胞淋巴组织 增生紊乱。 Rituximab对类风湿性关节炎、 免疫性血小板减少性紫癜、 免疫性溶血性 贫血、 系统性红斑狼疮和多发性硬化等自身免疫紊乱性疾病的作用也在研究中[7]。
3.2联合化疗治疗 B细胞淋巴瘤
徐志巧等 [8]应用同期对照的前瞻性研究方法，将 22例 B细胞性 NHL患者分为研 究组 (Rituximab组)和对照组， 研究组 11例用 CHOP方案 (环磷酰胺、 多柔比星、长春 新碱和泼尼松)联合 Rituximab治疗； 对照组 11例单用 CHOP方案。 结果 Rituximab 组完全缓解率 (CR)达 72.7% (8/11), 总有效率 90.9% (10/11)； 对照组 CR 为 36.4% (4/11), 总有效率为 54.6% (6/11)， 两组疗效差异有统计学意义。 初步研究结果提示， Rituximab联合 CHOP方案治疗 CD20阳性的 B细胞性 NHL的疗效显著， 不良反应 与单纯化疗相似， 可作为该病目前的首选方案。
3.3在风湿性疾病治疗中的应用
类风湿关节炎 (rheumatoid arthritis, RA)是一种以滑膜炎为主要病理改变的全身性 自身免疫病， 以慢性破坏性关节病变为特征。 B细胞是类风湿因子 (RF)、 抗瓜氨酸抗 体 (抗 CCP)等自身抗体的主要来源细胞， 同时研究表明 RA滑膜中 T细胞活化主要依 赖于 B细胞抗原呈递作用 [9]， 是 RA等自身免疫病发病机制核心环节之一。
Edwards等[1()]报道了 161例大样本的随机双盲对照研究，试验分为口服甲氨蝶吟 组(≥10 mg/周）、 ituximab组 (lg dl， dl5)、 Rituximab联用环磷酰胺组 (750mg d3， dl7)、 Rituximab联用甲氨蝶吟组， 结果显示， Rituximab治疗组 24周后达到 ACR20 65%〜 76%, 单用甲氨蝶吟治疗组为 38%， 两者比较有显著性差异 (PO.025); 持续观察 48 周， Rituximab治疗组疗效 (ACR20 33%〜65%)与单用甲胺蝶吟组 (ACR20 20%)相比仍 有显著性差异 (P≤0.01)。在治疗第 24周达 ACR50缓解的患者，单用 Rituximab组 (33%)、 联用甲氨蝶吟组 (43%)、 联用环磷酰胺组 (41%)均高于甲氨蝶吟组 (13%)。 此外， Rituximab治疗组患者的 RF水平迅速下降。 且 24周内维持较低水平。 而单用甲氨喋 吟组 RF水平仅一过性轻度下降。 该试验 Rituximab疗效与 B细胞清除情况相关， 证 明了 Rituximab 在 RA 中的治疗作用以及 B 细胞在 A发病中的重要作用。 针对 Rituximab对 RA的治疗作用的一系列研究也表明 Rituximab治疗 RA疗效稳定，是生 物治疗的新途径。
3.4对系统性红斑狼疮的治疗作用
系统性红斑狼疮 (； systemic lupus erythematosus, SLE)是多系统受累、 以多种自身抗 体表达的自身免疫性疾病。 B细胞在 SLE发病机制中发挥了关键性作用： 分泌大量 致病性自身抗体和白介素 10 (IL-10)、 白介素 6 (IL-6)、 肿瘤坏死因子 (TNF-γ)等多种 细胞因子； 高选择性的抗原呈递作用， 将抗原呈递给 T细胞， 同时剌激调节 T细胞、 树突状细胞活化[11]。
Looney等[12]报道了 17例 Rituximab治疗 SLE的 Ι/Π期扩大临床试验， 均为活动 性狼疮。 大部分患者同时接受免疫抑制剂治疗。 分为三组接受低、 中、 高不同剂量的 Rituximab, 分别为 100mg/m2、 275mg/m2、 375mg/m2每周 1次、 共 4周。 结果表明疾 病控制与 B细胞清除情况有关。 11例 B细胞清除 (CD19阳性细胞<5/1；1)患者皮疹、发 热、 关节痛等临床症状可缓解。系统性红斑狼疮活动评分 (SLAM)多在用药 2〜3个月 时缓解， 持续 12个月。 初步临床研究证明 Rituximab治疗 SLE是可行的，其疗效和安全性期待大样本临 床研究。
3.5 Rituximab治疗小儿免疫性血小板减少性紫癜
免疫性血小板减少性紫癜 (immunologic thrombocytopenic purpura, ITP)是一种自 身免疫性出血性疾病。 目前研究证明， ITP患者 T淋巴细胞调节紊乱， 导致 B淋巴细 胞功能紊乱， 产生针对自身血小板的 IgG抗体， 进而使血小板破坏增多。 文献报道 Rituximab可快速持久地清除循环中的 B淋巴细胞， 减少自身抗体的产生， 从而减少 血小板的破坏。 Taube等[13]使用 Rituximab治疗了 22例慢性 ITP患儿， 在 Rituximab 用后的 1〜2周外周血 CD20阳性 B淋巴细胞呈明显下降， 2〜3个月后逐渐恢复正 常。 结果 7例完全缓解， 6例部分缓解， 5例复发， 反应率为 59% (13/22)， 复发率为 38% (5/13), 持续缓解的时间是 2〜16个月。 石淑文等[14]用 Rituximab治疗了 3例小 儿慢性 /难治性 ITP， 3例均为联合用药， 1例联合阿赛松治疗部分缓解， 治疗后患儿 血小板持续维持相对较高的水平达 18个月之久，且不再需要 IVIG冲击治疗，阿赛松 逐渐减量至停用， 余两例分别联合了大剂量 IVIG、 琥珀酸氢化考的松和长春新碱， 治疗后血小板有短暂的回升 (分别是 21天和 11天)， 但很快又降至同入院前的低水平 且持续不见回升， 认为短暂的血小板回升可能是大剂量 IVIG、 琥珀酸氢化考的松或 长春新碱的作用，为不缓解病例。上述结果表明 Rituximab对 ITP有较好的治疗作用， 能够快速持久地清除循环中的 B 淋巴细胞， 减少自身抗体的产生， 从而减少血小板 的破坏，可作为临床用药。但部分病例这种清除是暂时的， 停用 Rituximab后循环中的 B淋巴细胞又会恢复， 这可能和部分病人再次复发有关。 关于 Rituximab 治疗慢性 / 难治性 ITP的总反应率成人为 23〜75% [15]， 儿童为 66%， 基本相同。
4.人源抗体的制备
20世纪 70年代中期杂交瘤技术问世， 由此制备出的单克隆抗体 (MAb)是抗单一 抗原决定簇的抗体， 具有高度的特异性和均一性， 且能大量制备， 这成为抗体研究领 域的一次重大革命。 1986年， 应用此技术制备的第一个单克隆抗体药物 OKT3被美 国 FDA批准上市， 被用于治疗器官移植后的免疫排斥。 但由于抗体是鼠源的， 在病 人中引发了严重的免疫反应， 这就大大阻碍了抗体药物的发展和利用。
随着抗体被用来开发成药物， 它的一些特征也将被严格考量， 像免疫原性、 亲和 力、 稳定性、 效应功能、 半衰期、 组织渗透性及其分布等[21]。 在鼠源 MAb生产变得 成熟时， 研究者便预想通过常规的杂交瘤技术生产人源 MAb， 但由于人杂交瘤细胞 系不能稳定的生产高产量的抗体，并且对于很多抗原来说，人的体内免疫是不可行的。 这样， 基因工程抗体由然而生， 1994年， 第一个嵌合抗体 ReoPro获准上市， 临床应 用表明其免疫原性远优于鼠源抗体，这就掀起了继杂交瘤单克隆抗体之后抗体工程研 究领域的又一次革命。 特别是 90年代中期以来， 为进一步降低免疫反应的发生， 多 种人源化改造技术被成功开发并得以应用，各种各样的基因工程抗体及抗体库不断出 现[23] [24]， 使得大规模生产医用人源化抗体成为可能。 从 1997年开始， 大批的单抗药 物被 FDA批准上市。
单抗人源化改造技术的应用打破了抗体药物产业化的瓶颈。迄今， 按照人源化程 度的高低大体可分为三种技术： 嵌合、人源化和全人源， 其基因中人源序列占比分别 为 75%、 95%和 100% [22]。 其中嵌合抗体是将鼠杂交瘤单克隆抗体基因的可变区和人 的恒定区连接在一起， 然后在哺乳动物细胞中进行表达产生； 而人源化抗体则是除了 将抗体的恒定区换成人源的之外， 更进一步的将可变区的 FR区转换成人源的[2()]， 从 而降低免疫原性； 而全人源抗体制备则是有四种途径，一种从采集的病人血样中构建 Fab或者 ScFv文库， 并通过噬菌体展示筛选抗体库， 来产生人源 MAb [18]。 2002年， 第一个全人源 MAb， Humira, 就是通过噬菌体展示开发出来的。 第二种生产人源抗 体的策略是用含有人免疫球蛋白基因位点的转基因小鼠， 进行免疫， 可以产生人抗体 的免疫应答， 这样通过传统的杂交瘤技术可以来生产人源 MAb[17]， 从转基因小鼠衍 生而来的人源 MAb正在临床试验中。 第三种途径是通过分离培养人外周血淋巴细胞 或扁桃体淋巴细胞， 然后用抗原进行免疫剌激， 产生免疫应答， 然后再和人类骨髓瘤 细胞融合产生杂交瘤细胞， 经筛选得到人源单克隆抗体， 并立即进行分子克隆抗体基 因， 使用其他可长期稳定表达的哺乳动物细胞来表达目的抗体， 这种抗体完全是在人 细胞环境中生成的。 第四类途径是通过分离和低密度 （约 500 B细胞每 96孔） 培养 人外周血淋巴细胞或扁桃体淋巴细胞， 对培养上清进行抗原特异性筛选， 对阳性孔 B 细胞进行分子克隆获得人抗体基因，再使用其他可长期稳定表达的哺乳动物细胞来表 达目的抗体。
本发明的全人源单克隆抗体就是基于上述第三种途径并加以随机突变筛选得来 的。 利用人-人杂交瘤产生的单克隆抗体被认为是在氨基酸序列和糖基化谱上都要优 于嵌合抗体和人源化抗体的全人源抗体。 但是由于难以找到使人 -人杂交瘤稳定传代 的融合伴侣细胞， 且由于融合效率低、 细胞培养困难及生产能力低等问题， 制备稳定 传代且能持续产生单克隆抗体的人-人杂交瘤细胞系一直存在较大难度， 且成功的实 例甚少。 本发明对表达人抗体的人-人杂交瘤快速进行包括特异性和功能学在内的筛 选，并将筛选出的杂交瘤细胞进行分子克隆化以保存抗体基因，从而克服了上述困难。
随着人源化和全人源抗体技术的成熟，如何提高抗体的亲和力成了目前所有基因 工程抗体药物需要解决的关键问题。这是因为增加一个抗体的亲和力将降低抗体药物 的剂量并起到更好的生物活性，还可能使其治疗更多的疾病及降低剂量相关的毒性[18] [22]。 此外， 费用也将大大降低。 多项独立的研究表明， 抗体亲和力和生物学活性在达 到极限值之前是成线性关系的。 围绕这一问题人们已经从不同的方向展开了研究， 提 高抗体亲和力的途径基本有两条途径， 其中一个途径是通过随机突变 CDR或是整个 的 VR， 然后从这个大量的突变体库里筛选更高亲和力的抗体 [19]; 另一个途径是通过 集中突变或是建模模拟体内亲和力成熟的热点区域， 建立一个小的突变体库[18] [19]。 综合起来说， 当进行体外抗体亲和力成熟时， 应考虑 4个主要方面： ①在抗体基因的 何处导人突变； ②如何导入突变； ③如何从较低亲和力的抗体中选择稀有的更高亲和 力抗体。 ④如何鉴别更高亲和力抗体。
目前已批准上市的三种针对 CD20的抗体均为 Biogen-Idec公司创制。其中 Zevalin 和 Bexxar为鼠源单抗， 并且主要是通过抗体介导放射性同位素到肿瘤部分， 发挥放 射性细胞毒直接杀伤肿瘤细胞。 由于放射毒性和人抗鼠抗体的产生等因素， 导致临床 疗效不佳， 适应症狭窄。 Rituxan (即 Rituximab)系人鼠嵌合型抗 CD20单克隆抗体， 其可通过 CDC、 ADCC、 诱导 CD20 细胞发生凋亡或直接抑制恶性 B细胞的增殖来 发挥其治疗效果且都获得了很好的疗效。但是由于其 25%鼠源部分， 更容易产生鼠源 性免疫原性，即人抗鼠抗体反应。这将会引起较多的输液反应， 同时也无法长期用药。 因为在病人血清中循环的人抗鼠抗体将会中和 Rituxan而使其疗效下降甚至失效。 此 外 Rituxan本身杀瘤疗效还存在着提高的空间， 例如其目前临床有效率 （CR+PR) 约 为 50%， 不能连续用药， 而且缓解期只有一年， 因此有必要发明新的抗体分子来提升 此类药物的疗效[29]。 本发明正是针对 Rituximab不足之处而进行的新分子创制： （1 ) 变鼠人嵌合抗体为全人源抗体（2)通过新药物的提升，使其针对其他瘤细胞的 CDC， ADCC和凋亡能力增强以提高杀瘤疗效。 发明内容
本发明提供抗 CD20的一组新抗体， 这些抗体为全人源抗体， 并且作为一种新药 物， 这些抗体的多项 CD20特异性的细胞毒功效比美国同类药物美罗华强， 具有比美 罗华相同或更好的药效。可用于治疗和诊断炎性疾病、 自身免疫性疾病、 细胞增殖病 症、 心血管病、 血液病， 尤其是非霍奇金氏淋巴瘤、 B细胞淋巴瘤、 风湿性和类风湿 性疾病、 系统性红斑狼疮、 免疫性血小板减少性紫癜和 /或多发性硬化症等疾病。
本发明的抗体的重链恒定区为人 IgGl重链的恒定区， 轻链恒定区为人 κ链的恒 定区，重链可变区的氨基酸序列为 Seq ID NO: 1、 Seq ID NO: 2 Seq ID NO: 3 Seq ID NO: 4和 Seq ID NO: 9中的一个，轻链可变区的氨基酸序列为 Seq ID NO: 5、 Seq ID NO: 6、 Seq ID NO: 7、 Seq ID NO: 8和 Seq ID NO: 10中的一个。
其中优选 11种抗体， 其重链可变区和轻链可变区的氨基酸序列分别为：
重链可变区 轻链可变区
1) Seq ID NO: 9 Seq ID NO: 10；
2) Seq ID NO: 4 Seq ID NO: 6；
3) Seq ID NO: 2 Seq ID NO: 7； 4) Seq ID NO: 4 Seq ID NO: 5；
5) Seq ID NO: 3 Seq ID NO: 5；
6) Seq ID NO: 1 Seq ID NO: 8；
7) Seq ID NO: 3 Seq ID NO: 6；
8) Seq ID NO: 2 Seq ID NO: 6；
9) Seq ID NO: 1 Seq ID NO: 5；
10) Seq ID NO: 4 Seq ID NO: 7；
11) Seq ID NO: 3 Seq ID NO: 8 o
其中， 优选的组合是 1)和 2)， 更优选的是 1)。
由于抗体的结合特性主要由互补决定区 (CDR)来决定， 因此， 本发明的抗体还包 括重链可变区包含 Seq ID NO: 1-4和 9的 CDR序列、轻链可变区包含 Seq ID NO: 5-8 和 10的 CDR序列的那些抗体。 上述 CDR序列详见 Seq ID NO: 11 -26。
而且，本发明的抗体并不限于人 IgG和人 κ链，本发明抗体的可变区以外的区域 可以是其他类型， 例如人 IgM等。
本领域技术人员将知晓的是，上述抗体中的保守性氨基酸替换并不会实质性地影 响抗体的亲和力和结构， 尤其是所述保守性替换发生在恒定区时。本领域技术人员还 知晓， 根据上述抗体的氨基酸序列可以设计出编码其的核苷酸序列， 并且针对不同的 表达宿主对该核苷酸序列进行优化。 为了治疗、 检测、 实验或其他目的， 本发明的抗 体可以与同位素、 免疫毒素和 /或化学药物共价连接， 还可以与固体介质、 半固体介 质偶联， 还可以使用本发明的抗体的功能性片段， 这在本领域中是已知的。
本发明提供了一种方法， 使用该方法制造全人源抗体并且作为一种新药物。 目前 尚未有抗体药物系使用本发明的技术方法成功创制的报道。该方法所克服的最重要的 技术难点是 （1 ) 人淋巴细胞体外有效免疫技术； （2) 人杂交瘤基因不稳定， 必须快 速进行包括特异性和功能学在内的筛选，并将筛选出的杂交瘤细胞进行分子克隆化以 保存抗体基因； （3 )对已筛选出具有一定药理学功效的人源抗体通过分子生物学抗体 工程技术进行进一步优化改造。
所述抗体的制造及相关检测方法如下：通过提取不同人的外周血淋巴细胞或者是 人扁桃体淋巴细胞， 体外混合后， 用抗原和具有佐剂功用的分子共培养， 使其产生针 对抗原的抗体， 通过与骨髓瘤细胞融合， 产生临时稳定的人杂交瘤， 培养上清中含有 其分泌的人源抗体。 通过细胞 ELISA (CD20阳性细胞和 CD20阴性细胞双筛选） 筛 选出 CD20阳性杂交瘤细胞生长孔， 再以相对抗原亲和力， 细胞毒功能测试（ADCC、 CDC和凋亡实验）综合评估筛选最好的 10个细胞生长孔， 并立即进行亚克隆， 筛选 出保持抗原特异性的单克隆细胞株。立即进行测序和分子克隆抗体基因到适当的表达 载体中， 并将质粒 DNA扩增纯化于 -8CTC永久保存。根据相对抗原亲和力， 细胞毒功 能测试（ADCC、 CDC和凋亡实验）综合评估筛选最好的 3个单克隆细胞株的抗体基 因，以此作为模板对其进行体外抗体亲和力成熟工程。我们以对 CDR1、 CDR2、 CD 3 区进行随机突变和定点突变相结合的方式建立抗体亲和力成熟突变文库。以抗原特异 性和相对亲和力排序对文库进行筛选。 将每轮最好的 5〜10 抗体的重链可变区 (VH) 和轻链可变区 (VL)进行全排列组合形成全长抗体。 使用瞬转、 培养和微量亲和纯化 (Protein A )制备少量纯化抗体， 以细胞毒功能测试（ADCC、 CDC和凋亡实验）进 行筛选鉴定。在经此筛选出来的一组有良好细胞毒功效的抗体分子， 再对这些抗体可 变区特别是 CDR区序列检索， 确定此组具备 CD20特异性的细胞毒功效的抗体为首 次发现的新分子。在此组新分子里有一个或一个以上的新抗体分子其（1 )多项 CD20 特异性的细胞毒功效比美罗华强； （2 )皮下移植瘤和生存期的试验结果显示其具有比 美国同类药物美罗华相同和更好的药效。
在确定本发明的抗体的氨基酸序列后，可以通过人工合成编码该抗体的多核苷酸 并选取适合的宿主进行有效地表达来制备该抗体， 这在本领域中是公知的。
除非特别说明， 在本文中" Rituxan"、 "Rituximab"和 "美罗华"可以互换使用。 附图说明
图 1〜图 3为 Raji细胞 CDC测试结果;其中图 1是对不同人杂交瘤上清液的 CDC 功能学筛选；图 2是对亲和力成熟后 CHO细胞表达纯化人抗体 H2L3、 H2L6、 H2L10、 H8L3和 H8L10的 CDC功能学筛选；图 3是对亲和力成熟后 CHO细胞表达纯化人抗 体 H8L15、 H11L6、 H11L10、 H19L3和 H19L15的 CDC功能学筛选;
图 4〜图 6为 Raji细胞 ADCC测试结果； 其中图 4是对不同的人杂交瘤上清液 的 ADCC功能学筛选；图 5是对亲和力成熟后 CHO细胞表达纯化人抗体 H2L3、H2L6、 H2L10、 H8L3和 H8L10的 ADCC功能学筛选； 图 6是对亲和力成熟后 CHO细胞表 达纯化人抗体 H8L15、 H11L6、 H11L10、 H19L3和 H19L15的 ADCC功能学筛选; 图 7. 源自细胞株 1.105.3的重链经突变得到的 H1〜H20根据亲和力的排序； 图
8. 源自细胞株 1.105.3的轻链经突变得到的 L1〜L20根据亲和力的排序；对应的结果 数据和 KD值参见表 6;
图 9. 经筛选的重链突变基因和轻链突变基因组合的根据亲和力的排序； 对应的 结果数据和 KD值参见表 7;
图 10显示了抗体 H2L10治疗人 B细胞淋巴瘤的小鼠 Raji细胞侵润瘤模型的药 效学试验结果， 是对小鼠体重和健康状态的观察。 静脉注射 107 Raji淋巴瘤细胞形成 小鼠淋巴瘤疾病模型。 分五组分别为 1.生理盐水组、 2.美罗华组、 3.H2L10 (；即本发明 的代表性抗体)低剂量组、 4.H2L10中剂量组和 5.H2L10高剂量组。 癌细胞静脉注射 后 7天开始给药。腹腔注射每周一次。 图中为各组动物体重变化。 生理盐水组动物自 第 16天后开始明显下降并呈肿瘤恶液质。美罗华组在第 37天开始出现体重明显下降， 总体呈亚健康状态直至第 50天试验结束。 而 H2L10高中低三组动物均呈健康状态， 体重正常增长。 此结果清楚表明 H2L10 和美罗华均对淋巴瘤动物有肯定的疗效， H2L10各组包括低剂量组疗效均优于美罗华。
图 11显示了抗体 H2L10治疗人 B细胞淋巴瘤的小鼠 Raji细胞侵润瘤模型的生存 期试验结果。 静脉注射 107 Raji淋巴瘤细胞形成小鼠侵润型淋巴瘤疾病模型。 分五组 分别为 1.生理盐水组、 2.美罗华组、 3.H2L10低剂量组、 4.H2L10中剂量组和 5.H2L10 高剂量组。癌细胞静脉注射后 7天开始给药。腹腔注射每周一次。 生理盐水组动物自 第 23天开始明出现死亡， 到第 34天全部死亡。 美罗华组和 H2L10高中低三组动物 均无死亡直至第 50天试验结束。但美罗华组在第 37天开始出现体重明显下降， 并总 体呈亚健康情况直至 50天试验结束。此结果清楚表明 H2L10和美罗华均可肯定地延 长淋巴瘤动物的生存时间。
图 12显示了抗体 H2L10治疗人 B细胞淋巴瘤小鼠 raji细胞原位移植瘤抑瘤实验 结果。 小鼠接种后观察肿瘤生长情况， 待肿瘤体积为 40-100 mm3左右时， 按瘤体积 大小进行筛选， 瘤体积过大及未成瘤者不予入选。 筛选后随机分为 3组： 1.生理盐水 组、 2.美罗华组、 3.H2L10。 给药剂量 5mg/kg， 腹腔注射每周一次。 试验结果清楚表 明美罗华和 H2L10均可以明显抑制 Raji细胞皮下移植瘤的生长， H2L10药效和美罗 华有同等药效。
图 13A和 13B为抗体 A137、 H2L10和美罗华对 Raji细胞的 ADCC测试结果。 图 14为抗体 A137和美罗华对 Raji细胞的 CDC测试结果。
图 15显示了 A137和美罗华的亲和力测试结果， 图中的表格显示出了其相关参 数 Bmax和 KD及置信区间。
图 16显示了 A137和美罗华的治疗人 B细胞淋巴瘤小鼠 raji细胞原位移植瘤的 结果。 每只小鼠静脉注射 107 个 Raji淋巴瘤细胞形成小鼠侵润型淋巴瘤疾病模型， 5 天后腹膜内注射给药。 分 3组小鼠进行实验 (；每组 8只)： A. 生理盐水对照组； B. 美 罗华组 (剂量为 1 mg/kg体重)； C. A137组 (剂量为 1 mg/kg体重)。
图 17显示了用 A137和美罗华的治疗人 B细胞淋巴瘤小鼠 raji细胞原位移植瘤 时的小鼠体重变化曲线。 每只小鼠静脉注射 107 个 Raji淋巴瘤细胞形成小鼠侵润型 淋巴瘤疾病模型， 5天后腹膜内注射给药。 分 4组小鼠进行实验 (；每组 8只)： A. 生理 盐水对照组； B. 美罗华高剂量组 (3 mg/kg体重)； C. 美罗华低剂量组 (1 mg/kg体重)； 0. 137组(1 mg/kg体重)。 具体实施方式
下文将通过具体的实施例来阐明本发明， 但应理解的是， 以下实施例并不限制本 发明的范围。
实施例
实施例 1: 杂交瘤和载体的制备
1. 抗原和淋巴细胞的准备及免疫
1.1抗原准备：
1.1.1 CD20蛋白
购买人 CD20 (Novusbio)抗原， 用 PBS稀释后， 用 0.22μιη针式无菌滤器过滤。 1.1.2 表达 CD20膜蛋白的 293F 细胞的制备
购买 CD20的 cDNA (Origene， SC101205), 并设计、 合成 PCR扩增引物： CD20正向弓 I物： 5 '-TCAGGAGTTTTGAGAGCAAAATG-3 '
CD20反向弓 I物： 5 '-AACAGAAGAAATCACTTAAGGAG-3 '
然后以 CD20的 cDNA为模板进行扩增、纯化，并克隆到 pCT .l载体（Invitrogen, K300001 )中， 测序验证后， 大量制备该质粒， 然后按照说明书转染 FreeStyle™ 293-F 细胞（Invitrogen, 790-07)， 48小时后，加入 lmg/ml的 G418 (Invitrogen, 10131035) 培养 2周， 稳定的 G418抗性克隆被筛选出来， 并冻存。
1.1.3抽提的 CD20膜蛋白
使用细胞膜蛋白与细胞浆蛋白抽提试剂盒 （购自碧云天生物研究所） 由 CD20-293F细胞依循试剂盒说明书抽提 CD20膜蛋白。用 0.22微米滤器过滤后分装于 1.5毫升细胞冻存管 -80°C保存。
1.2人淋巴细胞准备：
无菌摘取慢性扁桃体炎新鲜组织样本 (与徐州医学院附院耳鼻喉科合作， 扁桃体 取自扁桃体摘除手术的病人， 10例病人， 年龄 5〜10岁)， 用淋巴细胞分离液 (GE)分 离出其中的淋巴细胞， 取 30xl06个； 无菌抽取人新鲜外周血细胞， 用淋巴细胞分离 液 (GE)分离出其中的淋巴细胞， 取 30xl06个。
1.3 体外淋巴细胞免疫：
淋巴细胞培养基配方为： 1升 DMEM中含有 20%胎牛血清 (FBS)、 1% 的 200 毫 摩尔左旋谷氨酸 (Sigma, cat. # G 2150)、 1% 的 100x的非必须氨基酸 (Sigma, cat. # M 7145)、 1% 的 lOOx 青霉素链霉素混合液（Sigma, cat. # P 7539)、（10,000 U/ml penicillin + 10 mg/ml streptomycin) > 10 单位 /每毫升白介素 6 (Boehringer Mannheim, cat. # 1299972)、 20 单位 /每毫升人重组白介素 2 (R&D system, cat. # 202-IL-050)、 20 微克 / 每毫升美洲商陆有丝分裂原 （PWM) ( Sigma, cat. # L8777)、 1纳克 /毫升的无内毒素 CpG (定制合成） 和 1 支 OPI细胞培养添加剂 （Sigma, cat. # 0 5003)。
将 1.2中的两种来源的 16位个体淋巴细胞混合后在含 20%FBS的 RPMI-1640培 养基中进行混合培养， 淋巴细胞终浓度为 5xl05个 /ml， 同时加入下述步骤 2.1中获得 的小鼠腹腔伺养细胞 （终浓度为 lxlO5个 /ml)、 非必需氨基酸 （终浓度为 lug/ml)、 CD20抗原（蛋白免疫： 终浓度为 lug/ml， 细胞膜免疫组： 源于 106个 CD20-293细胞 的细胞膜蛋白 /ml)， 并放于二氧化碳培养箱中共培养 6-12天（第 5-6天换一次新鲜培 养液）。
2.杂交瘤细胞的制备
2.1伺养细胞的准备：
在融合前一天，断颈处死 BALB/c小鼠 (从北京维通利华实验动物技术有限公司购 买)， 浸泡于 75%酒精中 5分钟， 在超净台内， 无菌操作下用剪刀剪开腹部皮肤， 露 出腹膜，用注射器注入腹腔内 10 ml HAT培养基（20%FBS， HAT l x，其余为 DMEM)， 反复吹吸， 吸出培养基放入 50ml离心管内， 台盼蓝细胞计数， 调整细胞浓度为 2x l05 个 /ml， 加入 96孔板， 100 ul/孔， 放入二氧化碳培养箱中过夜。 此操作适用于所有需 要用到小鼠腹腔伺养细胞的实验。
2.2骨髓瘤细胞准备:骨髓瘤细胞 P3X63Ag8.653购自中科院上海生命科学研究院 细胞资源中心，将其培养至对数生长期， 1000 rpm下离心 5分钟，用不含 FBS的 DMEM 悬浮， 1000 rpm下离心 5分钟， 轻弹底部， 用不含 FBS的 DMEM培养基悬浮， 台盼 蓝计数， 细胞活性为 95%。
2.3细胞电融合及 HAT筛选：
收集与 CD20抗原共培养 6天后的淋巴细胞， 台盼蓝计数活细胞， OOrpm下离 心 5分钟， 弃上清， 轻弹底部， 用少量不含 FBS的 DMEM培养基悬浮； 与同样数量 的骨髓瘤细胞充分混匀， 补 DMEM培养基至 40ml， 2000rpm下离心 5分钟， 弃上清； 加入 2ml无菌 pronase溶液 （CalBiochem, 53702)， 轻微悬浮细胞团后， 作用 2 分钟， 加入 3-5ml FBS终止， 再加入电融合溶液至 40ml， 2000 rpm离心 5分钟， 弃 上清； 加入少量电融合溶液轻轻悬浮细胞团后， 补加至 40ml， 2000rpm离心 5分钟， 弃上清； 轻弹底部， 加入电融合溶液， 细胞计数， 调节细胞至 2xl06个 /ml， 分成 2ml/ 管； 按照 2ml体积的秒数进行设定， 启动电融合装置 （BTX); 融合后， lOOOrpm离 心 5分钟， 弃上清；加入少量 HAT培养基悬浮细胞后，加入适量体积的 HAT培养基， 按照融合时候的淋巴细胞加入量， lxlO6个 /板， 加入之前铺好伺养细胞的 96孔板中， 放入二氧化碳培养箱中进行培养。
融合后 7天， 对 HAT培养基进行 1/2换液， 融合后 10天， HAT培养基全换液。 2.4细胞上清抗原特异性 ELISA检测：
包被： 融合后 12天，用双套 ELISA法检测特异性抗体，一套为表达 CD20的 293 细胞膜包被，一套为未转染 293细胞膜包被; 4°C过夜包板，浓度为 50万个细胞膜 /50ul/ 孔， 包被液为 PH 8.3的 1%BSA PBS。 封闭： 用 PBST洗板 3次后以 10%脱脂牛奶粉 PBS封闭， 室温孵育 1小时； 然后用 PBST洗板 3次。 加样： 吸出 50ul细胞上清至孔 中， 阴性对照为 1:1000人血清 (健康成人捐献)， 阳性对照为 O.lug/ml美罗华注射液； 室温孵育 2小时后， 用 PBST洗板 3次。 二抗： 然后加入次辣酸根过氧化酶标记的羊 抗人 IgGl (Caltag), 50 ul/孔， 室温孵育 1小时后， 用 PBST洗板 3次。 显色： TMB 显色， 50ul/ml TMB， 10〜15分钟后以 2M 盐酸终止显色反应； A45。nm读数。
以 CD20-293包被板阳性同时 293包被板阴性孔为抗原特异性。
2.5扩大培养
第一次检测后， 选择阳性孔进行扩大至 4个 96孔， 过 2天， 对所扩孔进行检测， 确定阳性孔， 扩大至 24孔。 待 24孔细胞长至孔底约 1/3， 则进行 ELISA检测。 此方 法同样适用于后期抗体的检测。
2.6 细胞上清人抗体定量检测
对步骤 2.5产生的经再次 ELISA鉴定后仍为阳性的细胞孔培养上清，使用 Bethyl Laboratories的人 Ig 定量检测 ELISA试剂盒 （货号 E88-104), 具体步骤按试剂盒说 明书进行。
2.7 相对亲和力排序
样品准备： 将要比较的抗体， 即已知浓度的纯化抗体或由步骤 2.6定量检测后已 知所含抗体浓度的细胞培养上清， 稀释到 4-8个相同浓度 (120nM， 40nM, 13.1nM, 4.4nM)。然后按 2.4中描述的步骤进行 ELISA检测。 由于所有样品事先调整到相同抗 体浓度， OD值的差异将由抗体的亲和力引起。 OD值越高相对亲和力就越高。 使用 Prism软件作图和计算出相对每一抗体的亲和力。综合实施例 3〜5和此检验结果， 选 出抗原亲和力和功能学效能最高的 10个细胞生长孔， 即 1.1、 1.4、 1.6、 1.36、 1.72、 1.89、 1.105、 1.134、 1.146、 1.176。
2.8有限稀释法亚克隆、 细胞扩大培养
于亚克隆前 1天或者是当天采集小鼠腹腔伺养细胞 (用 HT培养基)， 细胞浓度为 lx lO5个 /ml，加入 96孔板， lOOul/孔。对经步骤 2.7检测后筛选的 1.1、 1.4、 1.6、 1.36、 1.72、 1.89、 1.105、 1.134、 1.146、 1.176阳性杂交瘤细胞株分别进行台盼蓝染色计数 后， 将细胞分别按以下浓度铺到板子中： 密度为 5个 /孔， 铺 1块； 1个 /孔， 铺 1块; 0.5个 /孔， 铺 2块。 放入二氧化碳培养箱中进行培养 5-7天后， 经倒置显微镜从孔底 观察细胞， 用记号笔圈下确认为单克隆的孔。 只转移这些单克隆孔的细胞培养上清， 50μ1/孔， 进行 ELISA检测 (见 2.4方法)。在经 ELISA确定抗原阳性的孔里， 根据细胞 状态和 OD值， 从被亚克隆的每杂交瘤细胞株 (1.1、 1.4、 1.6、 1.36、 1.72、 1.89、 1.105、 1.134、 1.146、 1.176)中选取最好的 3-5个单克隆细胞孔的上清进行下步骤 (2.9)鉴定。
2.9亚类的鉴定
包板羊抗人 IgG(Fc)， 2ug/ml， 4°C过夜， BSA封闭， 加入待测细胞上清， 37°C， 2小时， 加入酶标亚类二抗 IgGl， IgG2, IgG3 , IgG4, κ， λ ( Southern Biotechnology), 显色， A450读数， 判断所测细胞株的亚类为 IgGl， κ。 在加液前每步都用 PBST洗涤 3次。 从被亚克隆的每杂交瘤细胞株中由步骤 2.8筛选出的 3-5个单克隆细胞孔里， 选定一个确定为 IgGl-κ的单克隆细胞株， 其分别为： 1.1.81、 1.4.68、 1.6.14、 1.36.2、 1.72.108、 1.89.45、 1.105.3、 1.134.42、 1.146.78、 1.176.109。 将此 10个单克隆细胞株 扩大至 24孔（用含 20%FBS的 DMEM培养基）， 待细胞长好， 检测后， 扩大至方瓶。
2.10细胞冻存
冻存液配制： 10% DMSO， 60% FBS, 30% DMEM, 放于冰上预冷， 细胞吹悬后， 放入离心管中， 台盼蓝计数， 离心， 弃上清， 加入预冷冻存液， 按 2~5xl06个 /管， 进 行冻存。细胞放入程序降温盒（Nalgene)后置于 -70°C冰箱过夜， 第二天转入液氮中。
2.11测序、 克隆单克隆人杂交瘤基因
杂交瘤进行亚克隆后， 筛选出保持抗原特异性的单克隆细胞株 1.1.81、 1.4.68、 1.6.14、 1.36.2、 1.72.108、 1.89.45、 1.105.3、 1.134.42、 1.146.78、 1.176.109。 立即进 行测序和分子克隆， 抗体基因进入适当的表达载体， 并将质粒 DNA扩增纯化于 -80°C 永久保存。
实施例 2: 单克隆抗体制备及鉴定
1. 采用体外培养法制备抗体
将含有 15%胎牛血清的 DMEM培养基中生长的 1.1.81、 1.4.68、 1.6.14、 1.36.2、 1.72.108、 1.89.45、 1.105.3、 1.134.42、 1.146.78、 1.176.109细胞， 逐渐降低血清至无 血清或者是只含有少量血清的无血清培养基中（Invitrogen, 12338-026), 放入 225cm2 方瓶， 按接种 lx 105个 /ml的细胞量， 接种 100ml， 每株细胞各接种 4瓶。
2. 抗体纯化
取出在 225cm2方瓶培养了 10天左右的细胞上清，离心，去掉细胞碎片，经 rProtein A亲和层析进行抗体纯化。上样： pH7.4的细胞培养上清经 0.22um虑膜过滤后直接上 样； 流洗： pH7.4 PBS 流洗， 10倍的柱床体积； 洗脱： pH3.0甘氨酸溶液， 每段 0.5ml 收集洗脱液；读每段 OD280,将含有抗体的各段混合。再检测 OD280,按 1.580D=lmg 计算抗体浓度。 将收集的抗体溶液过滤 0.22um的滤器后无菌保存。 跑 SDS-PAGE胶 核实纯化抗体纯度。 做 LAL测试确定无内毒素污染 （Genscript, Cat: L00350), 将 此纯化抗体用于细胞毒功能测试 （见下述 ADCC、 CDC和凋亡实验）。
实施例 3: 补体依赖的细胞毒实验 （CDC)
Ramos aji和 Daudi细胞从 ATCC购买，培养基为 10% FBS, 1%丙酮酸钠和 1%
HEPES缓冲的 DMEM。 CellTiterGlo试剂盒购自 Promega (Madison, WI)。 正常人血清 由健康献血者全血分离获得。
按 lxlO5个 /孔， 将 Ramos、 Raji和 Daudi 细胞铺入 96孔板。 在 37°C 、 5% C02 的细胞培养箱中细胞和不同浓度的待测试抗体共孵育 10分钟， 其中待测试抗体为实 施例 2中得到的细胞上清抗体或实施例 6中得到的亲和力成熟抗体， 阳性对照为美罗 华， 阴性对照为纯化的人 IgG (；从健康人的血清纯化制备)。 然后加入正常人血清使其 在培养液中终浓度为 10%。 在 37°C 、 5% C02的细胞培养箱中， 将细胞、 不同浓度 的不同抗体与人血清共孵育 60分钟后， 通过 CellTiterGlo试剂盒测定细胞裂解率。如 为筛选杂交瘤培养上清， 则需要根据定量 ELISA 的上清中含抗体浓度的结果， 将要 筛选的众多上清抗体浓度调释至 l(^g/ml， 然后比较在相同浓度下各抗体的 CDC功 效， 以筛选出高 CDC功效的抗体杂交瘤抗体株。 使用 Prism软件作图和计算 EC50。 结果见图 1、 图 2、 图 3和表 1、 表 2。
表 1 : 对不同的人杂交瘤细胞株上清液的 CDC功能学筛选
Figure imgf000015_0001
表 2:对亲和力成熟后 CHO细胞表达纯化人抗体的 CDC功能学筛选 (表中排序抗体的 来源见实例 6)
Figure imgf000015_0002
实施例 4: 抗体依赖的细胞毒实验（ADCC)
靶细胞准备：用不含血清的 DMEM培养基，悬浮 Raji细胞至浓度为 1 <105个 /ml， 加入 Calcein AM (Sigma)至最终浓度为 10UM， 37°C， 孵育 50分钟。 离心去上清， 不 含血清的 DMEM培养基悬浮细胞至 104/75ul，铺到黑色透明底 96孔板中（Corning), 75ul/孔。 PBS 稀释待测抗体和阳性对照成不同浓度， 25ul/孔加入板子中， 室温孵育 30分钟。
效应细胞准备： 无菌采取新鲜正常人静脉血， 用抗凝剂使血液抗凝， 加入 osettesep Human NK cell Enrichment cocktail ( lml加入 50ul)， 加入与全血等体积的 含有 2%FBS的 PBS， 加入淋巴细胞分离液， 2150 rpm离心 30分钟； 转中间层细胞至 锥形管中， 补加含有 2%FBS的 PBS， 2150 rpm离心 10分钟， 弃上清； 用少量 PBS 悬浮细胞， 用台盼蓝计数， 调节细胞浓度至 105个 /75ul,
反应： 取 75ul的效应细胞至靶细胞和抗体的混合液中， 37°C孵育 4小时， 离心 板子， 1000 rpm离心 5分钟，转移上清至铺到黑色透明底 96孔板中（Coming), lOOul/ 孔， 板子放到酶标仪 （Luminescence Plate Reader) 读数。 结果例举见图 4、 图 5、 图 6和表 3、 表 4。 表 3 : 对不同的人杂交瘤细胞株上清液的 ADCC功能学筛选
Figure imgf000016_0001
表 4:对亲和力成熟后 CHO细胞表达纯化人抗体的 ADCC功能学筛选例举 (表中排序 抗体的来源见实例 6)
抗体 EC50 排序
H2L3 0.00967 6
H2L6 0.00847 4
H2L10 0.00235 1 H8L3 0.00907 5
H8L10 0.02351 9
H8L15 0.16059 10
H11L6 0.01034 7
H11L10 0.00401 3
H19L3 0.01053 8
H19L15 0.00369 2
ituximab 0.00273 实施例 5: 抗体诱导的肿瘤细胞凋亡实验
按 lxlO5个 /孔， 将 Ramos、 Raji和 Daudi 细胞铺入 96孔板。 在 37°C 、 5% C02 的细胞培养箱中细胞和不同浓度的待测试抗体 H2L10 (来自实施例 6)共孵育 48小时， 其中阳性对照为美罗华， 阴性对照为纯化的人 IgG。 48小时共孵育培养后， 取出 96 板，用 PBS洗涤 2次，以每孔 lOOul细胞培养液悬浮细胞，每孔加入 lul FITC-Annexin V (eBioscinece cat#: 88-8005 ) 并混合均匀， 室温避光染色 15分钟， 用 PBS洗涤 3 次。 以 200ul PBS悬浮细胞， 加样入细胞计数板， 在荧光显微镜计数细胞总数和染色 的凋亡细胞数求出细胞凋亡率。 结果见表 5。 结果显示： H2L10 在凋亡和 CDC实验 中， 药效比美罗华强约 2倍， 在 ADCC试验中， 与美罗华等效。
表 5: H2L10和美罗华之凋亡、 补体介导的细胞毒、 抗体介导细胞毒功效对比 (H2L10 来源见实例 6)。 细胞凋亡、 抗体介导细胞毒、 补体介导的细胞毒实验是三个主要的抗 癌药物药效学试验。 半数有效药物浓度 (EC5。)是比较不同药物药效学的标准指标。 表 中显示的实验是以美罗华为对照对 H2L10进行的背靠背药效学实验的结果。
Figure imgf000017_0001
实施例 6: 亲和力成熟
6.1 测序 Anti-CD20的重链和轻链
经免疫、 融合以及单克隆化后， 基于亲和力和细胞功能学实验结果选取抗 CD20 单克隆抗体细胞株 1.105.3为模板做抗体亲和力成熟实验。 抗体基因重链和轻链的序列分析。抗 CD20单克隆抗体细胞 1.105.3的总 RNA采 用 Invitrogen公司的 Trizol® reagent试剂盒（15596-026)进行提取； 接着采用 Takara 公司的 5 'RACE FULL试剂盒 （D315 ) 中的随机引物， 以总 RNA为模板， 反转录为 第一链 cDNA， 并按照试剂盒的说明书操作步骤， 在 5'端加上接头； 然后用试剂盒 自带的接头引物， 分别和人重链基因 IgGl恒定区特异引物以及轻链基因 κ链恒定区 特异性引物进行扩增得到抗 CD20单克隆抗体 (本文中称其为 "野生型"抗体)基因的 重链可变区和轻链可变区。 用于 PCR扩增的重链和轻链特异引物是：
IgGl 反向弓 I物： GCGCCTGAGTTCCACGACAC
κ链 反向弓 I物： CACAACAGAGGCAGTTCCAG
将 PCR扩增产物克隆到 T载体 (Takara, D101A)中， 并进行测序， 测序得到一致 的重链可变区和轻链可变区。
6.2 scFv的构建及 ELISA检测
为了提高该抗体的亲和力， 以构建的单链抗体 scFv为模板， 进行随机突变， 并 将突变的单链抗体进行噬菌体展示， 然后通过 ELISA筛选得到更高亲和力的抗体。 根据上一步测序结果， 设计重链可变区和轻链可变区特异性引物， 并分别在重链可变 区 5，端和 3，端加上 Sfil位点和 Xhol位点， 轻链可变区 5，端和 3，端加上 Nhel位点和 Notl位点， 重链可变区 (VH)和轻链可变区 (VL)扩增引物如下：
VH 反向引物： CCACCACTCGAGGC GCTCGAGACGGTGACCAGGGT
VL正向引物： TGGCGGGTCGACG GATATTGTGATGACCCAGACT
VL反向引物： TGTTCTGCGGCCGC TTTGATCTCCACCTTGGTC
分别以完全正确的 VH和 VL克隆为模板， PCR扩增重链可变区和轻链可变区后， 酶切， 分步克隆到噬菌体展示载体 pFL249 (基因合成构建)， 进行测序验证。
选择测序正确的克隆 A0026/pFL249-scFv转化到 TG1 感受态细胞 (Stratagene, 200123)中，挑取单克隆进行培养、诱导、收集上清， 以 Raji 细胞膜为抗原进行 ELISA 检测。
6.3 随机突变体库的构建、 筛选及测序分析
采用 Statgene公司的 GeneMorph II Random Mutagenesis试剂盒（200550)， 以 VH 正向引物和 VL反向引物进行易错 PCR扩增， 将扩增好的 PCR产物进行纯化、 酶切、 连接到 pFL249载体上， 然后电转化到 TG1感受态细胞中， 梯度稀释至适当的浓度， 涂布平板， 37°C过夜培养。
从单克隆密度适宜的平板上挑取单克隆到 96孔细菌培养板进行培养和诱导表达， 收集上清进行 ELISA检测，在 OD值比野生型高的单克隆中，根据 OD值从高到低选 择 200个单克隆进行测序。 分析测序结果， 去除 FR区突变的非人源克隆后， 根据 OD值， 从高到低， 分别 选择 20个突变的克隆的 VH和 VL用于构建全长抗体。
6.4构建全长抗体及亲和力排序
6.4.1 根据之前的结果， 设计重链可变区和轻链可变区特异性引物， 并分别在重 链可变区 5，端和 3，端加上 Sfil位点和 Sail位点， 轻链可变区 5，端和 3，端加上 Sfil位 点和 Bsiwl位点， 重链可变区和轻链可变区扩增引物如下：
VH 反向引物 1： GCCCTTGGTCGACGC GCTCGAGACGGTGACCAGGGT VL反向引物 1： AGCCACCGTACG TTTGATCTCCACCTTGGTC
分别以野生型的 VH和 VL克隆， 以及突变后的 20个 VH和 20个 VL克隆为模板， PCR扩增重链可变区和轻链可变区后， 酶切， 分别克隆到含有人的 IgGl恒定区和人 的 κ恒定区 pCI载体 (； Promega, E1731)中， 并进行测序验证。
6.4.2 分别将突变后的 20个全长重链 (H1〜H20)和野生型的全长轻链 (L)组合， 将 突变后的 20 个全长轻链 (L1〜L20)和野生型的重链 (H)组合， 将共 40 种组合采用 FreeStyle MAX转染系统 (Invitrogen, 16447-100) 共转染到 CHO-S细胞中， 37。C， 8% C02培养 6天后， 离心收集上清。
按前述实施例 1中 2.6步骤方法进行 40个抗体上清中抗体浓度定量，然后按前述 实施例 1中 2.7步骤方法进行相对亲和力排序。 结果如图 7、 图 8和表 6所示： 40个 突变体的上清的亲和力相比原始细胞株 1.105.3都有 10〜20倍不同程度的提高。分别 选定 5个亲和力最高的重链 H2、 H8、 Hll、 H16、 H19和 5个亲和力最高的轻链 L3、 L6、 L10、 L15、 L18。 表 6:突变后的重链和轻链的亲和力排序 (KD为解离常数 (单位： nM),是通过 Prism 软件以亲和力排序 ELISA数据计算得到的）
Figure imgf000020_0001
.4.3 将上一步筛选到的 5个重链突变体和 5条轻链突变体相互组合，配成 25个 全长突变抗体： H2L3, H2L6, H2L10, H2L15, H2L18, H8L3,H8L6, H8L10, H8L15,H8L18, H11L3, H11L6, H11L10, H11L15, H11L18, H16L3, H16L6, H16L10, H16L15, H16L18 H19L3, H19L3, H19L6, H19L19, H19L15, H19L18。 将它们重轻链共 转染， 培养、 收集上清， 再进行亲和力排序。
结果如图 9和表 7所示， 在 25个上清中， 有 10个 H/L组合比 Rituximab的亲和 力高。选择亲和力排序前 10的抗体， 即表 7中的前 10个抗体 H2L10、 H11L6、 H2L3、 H8L3、 H19L15、 H8L10、 H11L10、 H19L3、 H2L6和 H8L15, 按照实施例 3、 4对这 10个抗体进行 ADCC和 CDC测试， 测试结果见图 2、 图 3、 图 5、 图 6。 表 7: 突变后的 H/L组合的亲和力排序 (KD为解离常数 (单位： nM)， 是通过 Prism 软件以亲和力排序 ELISA数据计算得到的； Ritux表示美罗华）
Figure imgf000021_0001
6.5本发明的人抗体可变区序列
经最终的测序验证，表 7所示的前 10个抗体的重链恒定区为人 IgGl重链的恒定 轻链恒定区为人 κ链的恒定区， 重链可变区和轻链可变区的氨基酸序列如下： 6.5.1重链可变区序列及其互补决定区 (CDR)
Seq ID NO: 1为 H19的可变区的氨基酸序列
Seq ID NO: 2为 H11的可变区的氨基酸序列
Seq ID NO: 3为 H8的可变区的氨基酸序列
Seq ID NO: 4为 H2的可变区的氨基酸序列
Seq ID NO: 9为 A137H的可变区的氨基酸序列
Seq ID NO: 11为 H19 Hll H8和 A137H的 CDR1序列
Seq ID NO: 12为 H2的 CDR1序列
Seq ID NO: 13为 H19的 CDR2序列
Seq ID NO: 14为 H2 H11的 CDR2序列 Seq ID NO: 15为 A137H的 CDR2序列
Seq ID NO: 16为 H8的 CDR2序列
Seq ID NO: 17为 H19、 H2、 H8和 A137H的 CDR3序列
Seq ID NO: 18为 HI 1的 CDR3序列
6.5.2轻链可变区序列及其互补决定区 (CDR)
Seq ID NO: 5为 L3的可变区的氨基酸序列
Seq ID NO: 6为 L10的可变区的氨基酸序列
Seq ID NO: 7为 L6的可变区的氨基酸序列
Seq ID NO: 8为 L15的可变区的氨基酸序列
Seq ID NO: 10为 A137L的可变区的氨基酸序列
Seq ID NO: 19为 L3的 CDR1序列
Seq ID NO: 20为 L10的 CDR1序列
Seq ID NO: 21为 L6的 CDR1序列
Seq ID NO: 22为 L15的 CDR1序列
Seq ID NO: 23为 A137L的 CDR1序列
Seq ID NO: 24为 L3、 L6、 L10、 L15和 A137L的 CDR2序列
Seq ID NO: 25为 L3、 L6、 L10和 A137L的 CDR3序列
Seq ID NO: 26为 L15的 CDR3序列。
实施例 7: 动物体内药效试验
7.1 本发明的抗体 H2L10治疗人 B细胞淋巴瘤 Raji细胞侵润瘤模型的药效学试 验
7.1.1小鼠体重和健康状态的观察
静脉注射 107Raji淋巴瘤细胞形成小鼠淋巴瘤疾病模型。 分五组分别为 1.生理盐 水组 (无抗体)、 2.美罗华组 (单次剂量为 3.5mg/kg小鼠体重)、 3.H2L10低剂量组 (单次 剂量为 lmg/kg)、 4.H2L10中剂量组 (；单次剂量为 3.5mg/kg)和 5.H2L10高剂量组 (；单次 剂量为 10mg/kg)。 癌细胞静脉注射后 7天开始给药。 腹腔注射每周一次。 结果见图 10。
7.1.2小鼠体内生存期实验
静脉注射 107 Raji淋巴瘤细胞形成小鼠淋巴瘤疾病模型。 分五组分别为 1.生理盐 水组 (无抗体)、 2.美罗华组 (单次剂量为 3.5mg/kg)、 3.H2L10 低剂量组 (单次剂量为 lmg/kg)、 4.H2L10中剂量组 (；单次剂量为 3.5mg/kg)和 5.H2L10高剂量组 (；单次剂量为 10mg/kg)。 癌细胞静脉注射后 7天开始给药。 腹腔注射每周一次。 结果见图 11。
7.2 H2L10治疗人 B细胞淋巴瘤 Raji细胞原位移植瘤的抑瘤试验
小鼠接种人 B细胞淋巴瘤后观察肿瘤生长情况， 待肿瘤体积为 40-100 mm3左右 时，按瘤体积大小进行筛选，瘤体积过大及未成瘤者不予入选。筛选后随机分为 3组： 1.生理盐水组、 2.美罗华组、 3.H2L10, 其中美罗华组和 H2L10组的给药剂量均为 5 mg/kg小鼠体重。 腹腔注射每周一次。 实验结果见图 12。
实施例 8: 抗体 A137
重复进行实施例 1〜6的实验以筛选更多的符合本发明要求的抗体。 结果发现， 具有重链 A137H (其可变区序列为 SEQ ID NO: 9)和轻链 A137L (其可变区序列为 SEQ ID NO: 10)的全人源 CD20抗体 A137具有更好的效果。
具体而言， 在 ADCC测试中， A137的 EC5o小于美罗华 (；见图 13A)和 H2L10 (图 13B), 细胞杀伤效果更好； 在 CDC测试中， A137的效果与美罗华相似 (图 14)。 在亲 和力测试中， A137对 CD20的亲和力高于美罗华 (图 15)。
用 A137进行动物体内药效试验。注射了生理盐水的人 B细胞淋巴瘤模型小鼠在 约 21天时开始出现死亡， 至 40天时全部死亡； 而注射美罗华的小鼠组在约 44天时 开始出现死亡， 到约 130天时全部死亡； 相比之下， 注射 A137的小鼠组在约 47天时 开始死亡， 且到 140天以后仍有超过 20%的小鼠存活 (见图 16)。 这证明 A137的疗效 优于美罗华。 从图 17可以看出， 在小鼠体重维持方面， A137比 3倍剂量的美罗华的 效果更好： 到 150天时， 接受 A137的小鼠组的体重变化仍然缓慢， 且体重减少维持 在 15%以内； 而高剂量美罗华组小鼠在约 100天后体重下降就超过 15%， 在约 130 天时体重下降超过 35%。
本发明带来的有益效果
本发明正是针对 Rituximab不足之处而进行的新分子创制： （ 1 ) 变鼠人嵌合抗体 为全人源抗体 （2 ) 通过新药物的提升， 使其针对其他瘤细胞的 CDC， ADCC和凋亡 能力增强以提高杀瘤疗效。 （3 ) 治疗人 B细胞淋巴瘤 Raji细胞侵润瘤模型的药效学 试验和治疗人 B细胞淋巴瘤 Raji细胞原位移植瘤的小鼠药效学试验， 在生存期和抑 制肿瘤生长上均显示不低于或比美罗华更好的疗效。
本发明的抗体分子可作为药物候选分子将会对淋巴瘤和自身免疫性疾病有更好 的疗效， 有益于人类疾病的治疗。 参考文献
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Claims

权利要求书
1. 一种抗 CD20的全人源单克隆抗体， 所述抗体具有重链可变区和轻链可变区， 所述重链可变区和轻链可变区各自具有 3个互补决定区 (CDR)， 其中，
所述重链可变区的 CDR1的氨基酸序列为 Seq ID NO: 11或 Seq ID NO: 12， 所述重链可变区的 CDR2的氨基酸序列为 Seq ID NO: 13、 14、 15和 16中的一个， 所述重链可变区的 CDR3的氨基酸序列为 Seq ID NO: 17或 Seq ID NO: 18， 所述轻链可变区的 CDR1的氨基酸序列为 Seq ID NO: 19、 20、 21、 22和 23中的 一个，
所述轻链可变区的 CDR2的氨基酸序列为 Seq ID NO: 24， 并且
所述轻链可变区的 CDR3的氨基酸序列为 Seq ID NO: 25或 Seq ID NO: 26。
2. 如权利要求 1所述的抗体，所述抗体的重链可变区的氨基酸序列为 Seq ID NO: 1、 Seq ID NO: 2、 Seq ID NO: 3、 Seq ID NO: 4和 Seq ID NO: 9中的一个， 所述抗体 的轻链可变区的氨基酸序列为 Seq ID NO: 5、 Seq ID NO: 6、 Seq ID NO: 7、 Seq ID NO: 8和 Seq ID NO: 10中的一个。
3. 如权利要求 2所述的抗体， 其中所述抗体的重链可变区和轻链可变区为：
1) 氨基酸序列为 Seq ID NO: 9的重链可变区， 和氨基酸序列为 Seq ID NO: 10的 轻链可变区；
2) 氨基酸序列为 Seq ID NO: 4的重链可变区， 和氨基酸序列为 Seq ID NO: 6的 轻链可变区；
3) 氨基酸序列为 Seq ID NO: 2的重链可变区， 和氨基酸序列为 Seq ID NO: 7的 轻链可变区；
4) 氨基酸序列为 Seq ID NO: 4的重链可变区， 和氨基酸序列为 Seq ID NO: 5的 轻链可变区；
5) 氨基酸序列为 Seq ID NO: 3的重链可变区， 和氨基酸序列为 Seq ID NO: 5的 轻链可变区；
6) 氨基酸序列为 Seq ID NO: 1的重链可变区， 和氨基酸序列为 Seq ID NO: 8的 轻链可变区；
7) 氨基酸序列为 Seq ID NO: 3的重链可变区， 和氨基酸序列为 Seq ID NO: 6的 轻链可变区； 8) 氨基酸序列为 Seq ID NO: 2的重链可变区， 和氨基酸序列为 Seq ID NO: 6的 轻链可变区；
9) 氨基酸序列为 Seq ID NO: 1的重链可变区， 和氨基酸序列为 Seq ID NO: 5的 轻链可变区；
10) 氨基酸序列为 Seq ID NO: 4的重链可变区， 和氨基酸序列为 Seq ID NO: 7的 轻链可变区； 或
11) 氨基酸序列为 Seq ID NO: 3的重链可变区， 和氨基酸序列为 Seq ID NO: 8的 轻链可变区。
4. 如权利要求 1〜3中任一项所述的抗体， 所述抗体的重链恒定区为人 IgGl重 链的恒定区， 所述抗体的轻链恒定区为人 κ链的恒定区。
5. 如权利要求 1〜4中任一项所述的抗体， 所述抗体在其可变区以外的区域的氨 基酸序列中具有一个或多个保守性突变。
6. 一种多核苷酸， 所述多核苷酸编码权利要求 1〜5中任一项所述的抗体。
7. 一种载体， 所述载体中包含权利要求 6所述的多核苷酸。
8 一种抗 CD20的结合物，所述结合物包含与同位素、免疫毒素和 /或化学药物共 价连接的权利要求 1〜5中任一项所述的抗体。
9. 一种偶联物， 所述偶联物为权利要求 1〜5中任一项所述的抗体或权利要求 8 所述的结合物与固体介质或半固体介质偶联而形成的。
10. 权利要求 1〜5中任一项所述的抗体、权利要求 8所述的结合物或权利要求 9 所述的偶联物在制备用于治疗下述疾病的药物中的应用， 所述疾病选自炎性疾病、 自 身免疫性疾病、 细胞增殖病症、 心血管病、 血液病， 进一步的， 为： 非霍奇金氏淋巴 瘤、 B细胞淋巴瘤、 风湿性和类风湿性疾病、 系统性红斑狼疮、 免疫性血小板减少性 紫癜和 /或多发性硬化症。
11. 一种药物组合物， 所述药物组合物包含权利要求 1〜5中任一项所述的抗体、 权利要求 8所述的结合物和 /或权利要求 9所述的偶联物。
12. 一种用于诊断炎性疾病、 自身免疫性疾病、 细胞增殖病症、 心血管病、 血液 病， 尤其是非霍奇金氏淋巴瘤、 B细胞淋巴瘤、 风湿性和类风湿性疾病、 系统性红斑 狼疮、 免疫性血小板减少性紫癜和 /或多发性硬化症的试剂盒， 所述试剂盒包含权利 要求 1〜5中任一项所述的抗体、 权利要求 8所述的结合物和 /或权利要求 9所述的偶 联物。
13. 一种治疗炎性疾病、 自身免疫性疾病、 细胞增殖病症、 心血管病、 血液病， 尤其是非霍奇金氏淋巴瘤、 B细胞淋巴瘤、风湿性和类风湿性疾病、系统性红斑狼疮、 免疫性血小板减少性紫癜和 /或多发性硬化症的方法， 所述方法包括向有需要的患者 施用治疗有效量的权利要求 1〜5中任一项所述的抗体、 权利要求 8所述的结合物、 权利要求 9所述的偶联物和 /或权利要求 11所述的药物组合物。
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