WO2016019726A1 - 低免疫原性抗TNF-α全人源单抗及其应用 - Google Patents

低免疫原性抗TNF-α全人源单抗及其应用 Download PDF

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WO2016019726A1
WO2016019726A1 PCT/CN2015/074528 CN2015074528W WO2016019726A1 WO 2016019726 A1 WO2016019726 A1 WO 2016019726A1 CN 2015074528 W CN2015074528 W CN 2015074528W WO 2016019726 A1 WO2016019726 A1 WO 2016019726A1
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tnf
seq
antibody
variable region
chain variable
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孙乐
张小刚
李茂华
张翠娟
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Abmax Biotechnology Co Ltd
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Abmax Biotechnology Co Ltd
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Definitions

  • the invention relates to the preparation and application of a human genetically engineered antibody for treatment, and mainly relates to an antibody specific for human TNF- ⁇ and an application thereof.
  • TNF- ⁇ is a cytokine that naturally occurs in inflammation and immune responses. The study found that TNF- ⁇ levels are elevated in the synovial fluid of patients with rheumatoid arthritis (RA) and play an important role in pathological inflammation and joint destruction.
  • monoclonal IgG antibodies or soluble TNF- ⁇ receptors are generally used to neutralize TNF- ⁇ in vivo.
  • Commercially available monoclonal antibodies are as follows: Infliximab is a humanized TNF- ⁇ murine monoclonal antibody, and etanercept is a fusion protein conjugated to the Fc end by two human TNF- ⁇ receptors (p75).
  • Binding ability assay using radiolabeled TNF- ⁇ showed that infliximab binds to monomeric (inactive) and trimer (active) types of soluble TNF; etanercept is more prone to binding active In the form of a polymer, TNF- ⁇ and TNF- ⁇ .
  • Adalimumab monoclonal antibody is a fully human TNF- ⁇ monoclonal antibody developed by Abbott Laboratories of the United States that specifically binds to TNF and blocks its interaction with TNF receptors on the surface of p55 and p75 cells.
  • adalimumab monoclonal antibody is more immunogenic in the host, and up to 26% of patients use the monoclonal antibody to develop an immune response, which brings risks and inconvenience to the use of clinical patients, and the drug half-life is relatively short, using In the process, it is also necessary to increase the dose of the drug to make up for the defects in this aspect.
  • the immunogenicity of the antibody drug can be reduced by replacing the highly immunogenic non-antigen binding site in the antibody with a low immunogenic homologous sequence without affecting the affinity and specificity of the antibody.
  • the safety of the monoclonal antibody can be improved; on the other hand, the half-life of the drug can be increased, and the dose can be reduced while also improving the therapeutic effect.
  • a first object of the present invention is to provide a monoclonal antibody which is low in immunogenicity and which is fully human-derived against TNF- ⁇ .
  • Another object of the present invention is to provide a preparation of low immunogenic whole human anti-TNF- ⁇ . Monoclonal antibody method.
  • the present invention evaluated the original sequence of adalimumab (purchased from Abbott Laboratories, USA) using commercial DNAStarTM software and found that the adalima immunogenicity coefficient was 16.
  • the non-antigen-binding fragment (FR) in the variable region of the antibody was evaluated for immunogenicity using the above software to find a highly immunogenic related sequence.
  • all FR segments involved in the light and heavy chains in the human antibody gene pool were searched, and some relatively low immunogenic segments were selected by comparison.
  • the corresponding changes in the segment were replaced with the corresponding segments in adalimumab, followed by 3D modeling using Pymol protein molecular simulation software, and the sequence maintaining the original antigen binding site was selected for gene synthesis. , sequencing, and selecting the correct sequence for sequencing.
  • the low immunogenic anti-TNF- ⁇ full human monoclonal antibody of the present invention is an amino acid modification in the FR region of the light chain variable region and the heavy chain variable region of the original adalima sequence, thereby reducing the immunogenicity thereof.
  • the amino acid sequences of the light chain variable region and the heavy chain variable region of the original adalim sequence are shown in SEQ ID NO. 23, 24, respectively.
  • the low immunogenic anti-TNF- ⁇ full human monoclonal antibody provided by the present invention has the light chain variable region amino acid sequence of any one of SEQ ID NO. 11 to 15 or SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region thereof is shown in any one of SEQ ID NOS. 16 to 20.
  • the low immunogenic anti-TNF- ⁇ full human monoclonal antibody provided by the present invention has the amino acid sequence of the light chain variable region as shown in SEQ ID NO. 13, and the amino acid sequence of the heavy chain variable region thereof. As shown in SEQ ID NO.
  • the nucleotide sequences encoding the same are shown in SEQ ID NO. 3 and SEQ ID NO. 9, respectively.
  • the human-derived anti-human TNF- ⁇ monoclonal antibody provided by the present invention has a light chain variable region having any light chain variable represented by the five base sequences of L1 to L5 (SEQ ID NOS 1-5).
  • the region sequence, the heavy chain variable region has any of the heavy chain variable region sequences represented by the five base sequences of h1 to h5 (SEQ ID NOS. 6 to 10).
  • the present invention screens 10 monoclonal antibody sequences which retain both the original antigen binding site and the affinity and original binding to human TNF- ⁇ .
  • the adalimumab antibodies are similar and specifically block the binding of TNF- ⁇ to the cell surface TNF receptors p55 and p75.
  • the present invention provides a coding gene for the light chain variable region and the heavy chain variable region of the above 10 monoclonal antibodies.
  • the 10 low immunogenic whole human anti-TNF- ⁇ monoclonal antibodies obtained by the invention are L3h2, L3h4, L5h2, L4h1, L4h2, L4h4, L1h3, L2h1, L0h4, L2h5, respectively.
  • nucleotide sequence of the light chain of the L3h2 mAb is as described in SEQ ID NO. 3
  • nucleotide sequence of the heavy chain is as described in SEQ ID NO. 7
  • amino acid sequences of the light chain are respectively SEQ ID NO.
  • amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L3h4 mAb is as described in SEQ ID NO. 3
  • nucleotide sequence of the heavy chain is as described in SEQ ID NO. 9
  • amino acid sequence of the light chain is SEQ ID NO.
  • amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L5h2 mAb is as described in SEQ ID NO. 5
  • nucleotide sequence of the heavy chain is as described in SEQ ID NO. 7
  • amino acid sequences of the light chain are respectively SEQ ID NO.
  • amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L4h1 mAb is as described in SEQ ID NO. 4, the nucleotide sequence of the heavy chain is as described in SEQ ID NO. 6, and the amino acid sequence of the light chain is SEQ ID NO, respectively. As described in .14, the amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L4h2 mAb is as described in SEQ ID NO. 4, the nucleotide sequence of the heavy chain is as described in SEQ ID NO. 7, and the amino acid sequences of the light chain are respectively SEQ ID NO. As described in .14, the amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L4h4 mAb is as described in SEQ ID NO. 4, the nucleotide sequence of the heavy chain is as described in SEQ ID NO. 9, and the amino acid sequence of the light chain is SEQ ID NO. As described in .14, the amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L1h3 mAb is as described in SEQ ID NO. 1
  • nucleotide sequence of the heavy chain is as described in SEQ ID NO. 8, respectively, and the amino acid sequences of the light chain thereof are respectively SEQ ID NO.
  • amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L2h1 mAb is as described in SEQ ID NO. 2, and the heavy chain
  • the nucleotide sequences are as described in SEQ ID NO. 6, respectively, the amino acid sequences of the light chains are as described in SEQ ID NO. 12, and the amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L2h5 mAb is as described in SEQ ID NO. 2
  • nucleotide sequence of the heavy chain is as described in SEQ ID NO. 10
  • amino acid sequence of the light chain is SEQ ID NO, respectively.
  • amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • nucleotide sequence of the light chain of the L0h4 mAb is as described in SEQ ID NO. 21
  • nucleotide sequence of the heavy chain is as described in SEQ ID NO. 9
  • amino acid sequence of the light chain is SEQ ID NO.
  • amino acid sequences of the heavy chains are as described in SEQ ID NO.
  • the present invention provides an expression vector for a monoclonal antibody light chain variable region and a heavy chain variable region gene comprising all of the above 10 low immunogenic fully human anti-TNF- ⁇ .
  • Host bacteria, host cells or expression cassettes containing the expression vector are also within the scope of the invention.
  • the present invention provides the use of the above 10 low immunogenic whole human anti-TNF- ⁇ monoclonal antibodies for the preparation of a medicament for treating autoimmune diseases.
  • the disease is rheumatoid arthritis, lupus erythematosus.
  • the present invention provides the use of the above-mentioned fully human anti-TNF- ⁇ monoclonal antibody for the preparation of a medicament for treating diseases with TNF- ⁇ as a target.
  • the drug is an antitumor drug, an anti-inflammatory drug or a drug for treating an autoimmune disease.
  • the present invention provides a drug or a detection reagent comprising the above-described fully human anti-TNF- ⁇ monoclonal antibody.
  • the invention provides a method for preparing the above-mentioned whole human anti-TNF- ⁇ monoclonal antibody, comprising the following steps: (1) analyzing the FR sequence of the human antibody in the NCBI database (see Figures 2 and 3 for details), according to the above As a result, the immunogenicity of the four non-antigen-binding fragments (FR) in the variable region of the antibody was evaluated by commercial DNAstar software to find a highly immunogenic fragment. Then, by finding the FR3 segments of all heavy chains in the known human antibody gene pool, some corresponding segments with low immunogenicity were selected. The corresponding heavy and light chain segments on adalimumab were replaced, and then 3D modeling was performed using Pymol protein molecular simulation software. It was found that most of the replacement alignment resulted in a large conformational change in the antigen binding site of the antibody, but There are also some parts that basically maintain the original antigen binding site;
  • the present invention performs whole-gene synthesis on the corresponding modified sequence,
  • the synthesized gene was sequenced and the correct sequence was selected for the next step.
  • the light chain variable region was designed to be cleavage site Kpn I+BamH I, and the heavy chain variable region was designed to be KpnI+AgeI.
  • the expression vector pJH16 vector was ligated, and Escherichia coli DH5 ⁇ was simultaneously transformed to obtain a heavy chain and light chain chimeric antibody expression vector. The results are shown in FIG. 1 . Simultaneous sequencing and sequence comparison of the constructed antibodies;
  • the present invention selects a corresponding combination to construct a stable strain by electroporation, and simultaneously uses MTX to screen the degree of antibody expression, and performs monoclonal screening on the stabilized strain after pressurization, and finally selects antibody production. Higher stable strain for use in subsequent experiments;
  • the monoclonal antibody produced by the present invention has a lower immune response in mice than adalimumab, and the half-life is prolonged.
  • the fully human anti-TNF- ⁇ antibody and adalimumab provided by the present invention are directed to the same site of TNF- ⁇ , but the immunogenicity and antibody conformation are different from adalimumab, and the drug half-life is prolonged compared with adalimumab, which is expected to be very desirable.
  • Biologically targeted therapeutic antibodies By immunogenic modification of adalimumab monoclonal antibody, the partial amino acid sequence of the antibody is altered, which will significantly reduce the risk of immunogenicity of the antibody drug in the patient.
  • the present invention shows that the improved adalimumab antibody of the present invention binds to TNF- ⁇ with similar affinity to the original adalimumab, and prolongs the half-life of the antibody drug and improves the therapeutic effect.
  • Figure 1 shows the results of restriction enzyme digestion of plasmid and synthetic gene; wherein a is the double digestion result of pJH16 plasmid (kpn I and Age I), 1: the double digestion result of plasmid.
  • Figure b shows the results of double digestion of pJH16 plasmid (kpn I and BamH I), 1: double digestion of plasmid.
  • Figure c shows the results of double digestion of the adamus primary heavy chain (kpn I and Age I) and light chain (Kpn I+BamH I) genes, 1: heavy chain double digestion results, 2: Light chain double digestion results; where M is DL 15000 marker.
  • Figure 2 is a BLAST alignment of the humanized light chain amino acid sequence; wherein, Figures 3a, 2b, and 2c are all BLAST alignment results.
  • Figure 3 is a BLAST alignment result of the humanized heavy chain amino acid sequence; wherein, the three graphs of Figures 3a, 3b and 3c are the results of the BLAST alignment.
  • Figure 4 adalimumab improved affinity EC 50.
  • Figure 5 Improved adalimin inhibits TNF- ⁇ -induced L929 cytotoxicity assay.
  • Figure 5a represents TNF- ⁇ -induced L929 cytotoxicity assay as a positive control for the experiment;
  • Figure 5b represents that the modified adalim antibody inhibits TNF- ⁇ -induced L929 cytotoxicity.
  • the immunogenicity-associated sequence was found by immunogenicity evaluation of the non-antigen-binding fragment (FR) in the variable region of the antibody by using the above software. Subsequently, all FR segments involved in the light and heavy chains in the human antibody gene pool were searched, and some relatively low immunogenic segments were selected by comparison. Based on the above information, the correspondingly changed parts of the segment are replaced with the corresponding segments of adalimumab, and the initially obtained human-derived anti-human TNF- ⁇ monoclonal antibody has its light chain variable region selection. Any base from L1 to L5, L0 (SEQ ID NO. 1 to 5, SEQ ID NO. 21) The light chain variable region sequence consisting of a sequence, the heavy chain variable region is selected from the heavy chain variable region sequence consisting of any one of the base sequences represented by h1 to h5 (SEQ ID NOS. 6 to 10).
  • the restriction enzyme sites on both sides of the light chain sequence were designed as Kpn I+BamHI, and the enzyme cleavage sites on both sides of the heavy chain sequence were designed.
  • the point is Kpn I+AgeI, and the above sequence was sent to Jinweizhi Company to synthesize the whole gene sequence, and the ligation vector used for the synthesis was pUC57.
  • the synthetic genes were returned, and they were digested with the sequence synthesized by Jinweizhi Co., respectively (see Fig. 1), and ligated overnight at 16 °C.
  • the target gene and the expression vector (pJH16) obtained by digestion were separated by agarose gel electrophoresis, and then the target band was excised and recovered by Qiagen Gel Extraction Kit, and ligated overnight according to the T4 DNA ligation system, and then transformed into Escherichia coli DH5 ⁇ , correspondingly.
  • the strains were subjected to plasmid extraction and sequencing, and the sequencing results showed that the sequences were identical, indicating that the antibody expression vector was successfully constructed.
  • E. coli DH5a was transfected with the pJH16 heavy chain, light chain expression vector constructed in Example 2.
  • the cells were inoculated in 100 ml of LB medium and cultured according to a conventional method.
  • the culture was harvested and the plasmid DNA was purified using Qiagen's UltraPure Plasmid DNA homozygous kit.
  • the purified plasmid DNA was transfected into 293F cells using the liposome assay kit of Invitrogen, and the procedure was carried out according to the manufacturer's instructions.
  • 293F cells were transfected with different light and heavy chain plasmid combinations.
  • the combination is shown in Table 1.
  • a total of 31 groups of 293F were transiently expressed, and a combination of reduced immunogenicity was selected from these 31 groups.
  • the culture supernatant was taken for detection of antibody expression, and the results are shown in Table 1 below:
  • L0h1 refers to the combination of the original adalo light chain variable region and the modified adalim heavy chain variable region h1 of the present invention, and so on.
  • Adh0l0 is the original adamu combination
  • CHO purchased from American Handsome Company
  • MTX pressure screening (MTX was purchased from Sigma) was performed using selective medium Opti-CHO (MTX-opi-cho medium).
  • the five gradients of 50, 100, 200, 400, and 800 nM were pressurized, and the cells after each round of compression were subjected to 7d titer measurement.
  • the method of determination was to determine the engineering cell strain after each step of pressurization by ELISA-double-anti-sandwich method. Antibody production.
  • the monoclonal screening was performed by limiting dilution method, which was mainly carried out by diluting the cells (96-well plate), and culturing at 37 ° C under 5% CO 2 for about 14 days, and taking 50 ⁇ l for antibody production.
  • the primary screening using ELISA-double-antibody sandwich assay is better for cloning and enrichment of the screening results.
  • the purification of the antibody was performed by using Protein A from GE.
  • the 200nM pressurized group was used to prepare the antibody stable strain.
  • the culture supernatant of the above 11 stable cell lines was directly isolated and purified by Protein A affinity chromatography column.
  • Human monoclonal antibodies of the invention (Note: 10 of them are modified and 1 is the original adalimumab).
  • the experimental process is detailed in the GE company's instructions.
  • the product is quantitatively detected by an ultraviolet spectrophotometer. The calculation formula is as follows:
  • This section uses the ELISA indirect method to measure antibody EC50 to assess antibody affinity.
  • TNF-a was diluted with PBS to 0.3 ug/ml; the diluted antigen was added to a 96-well plate at 100 ul/well, capped at 4 ° C overnight; the liquid in the well was removed, and washed with PBS.
  • the specific experimental procedure was as follows: rhTNF ⁇ , rhTNF ⁇ , rIFN ⁇ (recombinant human interferon ⁇ ), IL-1 ⁇ , IL-1 ⁇ , IL-2, IL-4 and IL-8 were diluted with PBS to 1 ug/ml; A good antigen 100 ul / well was added to a 96-well plate, and capped, overnight at 4 ° C; the liquid in the well was removed, washed three times with PBS, 200 ul / well, manually patted; closed with 5% milk 200 ul / well, closed 1h, tap the microplate every 15min to promote the reaction; remove the liquid in the well, wash once with PBS, 200ul/well, manually pat dry; add different light chain heavy chain combination antibody, 5% milk diluted, 100ul/ Wells, incubate for 1 h, tap the plate every 15 min to promote the reaction; remove the liquid in the well, wash three times with PBS, 200 ul / well, manually pat dry; add goat anti
  • This section uses the CCK-8 kit for the corresponding experiments.
  • This experiment mainly uses mouse fibroblast cell line L929 for apoptosis detection. The specific steps are as follows:
  • the control included a negative control and a positive control.
  • the negative control only added RPMI-1640 and cells.
  • the positive control only added rhTNF ⁇ and cells, and the rhTNF ⁇ concentration ranged from 2 ng/ml to 8.2 pg/ml.
  • the plates were incubated for 1-4 h in a 37 ° C incubator.
  • the absorbance at 470 nm was measured with a microplate reader.
  • OD value is not measured for a while, 10 ⁇ L of 0.1 M HCL solution or 1% w/v SDS solution may be added to each well, and the culture plate is covered and stored at room temperature in the dark. The absorbance does not change within 24 hours.
  • Basal immunization The antigen was mixed with an equal volume of Freund's complete adjuvant and fully emulsified, subcutaneously injected at a sub-point, and each Balb/c mouse was injected in an amount of 100 ⁇ g per injection.
  • Biodistribution of antibodies Sixteen tumor-bearing nude mice were randomly divided into 4 groups, 4 in each group. 563.5kBq (100 ⁇ L, 2.5 ⁇ g) of 125 I-antibody was injected from the tail vein, and the nude mice were sacrificed by the neck of the group at 2, 4, 8 and 24 h after injection, and blood and main organs and tissues were taken. The radioactivity count was weighed and the percent injection dose rate (%ID/g) of each organ or tissue was calculated.
  • Pharmacokinetic distribution test Take 5 BALB/c mice, each of which was injected with 100 ⁇ L of 125 I-antibody (370 kBq, 2 ⁇ g) via the tail vein at different time points after injection (5, 12, 30 min, 1, 2, 4). , 8, 11, 22, 34, 48, 72h) Blood was taken from the eyelids, weighed and measured by gamma counter, and the dose rate per gram of blood (%ID/g) was calculated. Analysis was performed using Grap h Prism software to calculate pharmacokinetic parameters. As a result, it was found that the 10 modified adalimumab of the present invention has a longer half-life than the original adalimumab (Fig. 6).
  • the low immunogenic anti-TNF- ⁇ full human monoclonal antibody provided by the present invention binds to human TNF- ⁇ with similar affinity to the original adalimumab antibody, and specifically blocks TNF- ⁇ and cell surface TNF receptors p55 and p75. Combination of.
  • the low immunogenic anti-TNF- ⁇ whole human monoclonal antibody of the present invention can be used for preparing a prophylactic and therapeutic antibody drug against tumor and other diseases such as inflammation and autoimmune diseases against a TNF- ⁇ target, and the antibody drug is expected to be It reduces the risk of producing immunogenicity in the patient, prolongs the half-life of the antibody, improves the curative effect, and has excellent clinical application value.

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Abstract

本发明提供了一种低免疫原性的抗TNF-α全人源单抗及其应用。通过对阿达木（adalimumab）单抗氨基酸序列分析，对其中免疫原性高的非抗原结合序列进行改进，构建并筛选出多个免疫原性有效降低的抗TNF-α全人源单抗，其与人TNF-α结合的亲和力和原始adalimumab抗体相近，可特异性地阻断TNF-α与细胞表面TNF受体p55和p75的结合。

Description

低免疫原性抗TNF-α全人源单抗及其应用 技术领域
本发明涉及治疗用人源基因工程抗体的制备及应用，主要是涉及特异性针对人TNF-α的抗体及其应用。
背景技术
TNF-α是一种在炎症和免疫应答中自然出现的细胞因子。研究发现在类风湿性关节炎(RA)患者的滑膜液中，TNF-α水平升高，并在病理性炎症和关节破坏方面起重要作用。目前一般采用单克隆IgG抗体或者可溶性的TNF-α受体来中和体内的TNF-α。市售的单抗有如下：英夫利昔是人源化的TNF-α鼠单克隆抗体，依那西普是由两条人TNF-α受体(p75)偶联到Fc端的融合蛋白。利用放射性标记的TNF-α进行结合能力测定，结果显示英夫利昔能结合单体(无活性)及三聚体(有活性)类型的可溶TNF；依那西普更倾向于结合有活性的聚体形式的TNF-α及TNF-β。阿达木(adalimumab)单抗是美国雅培公司研制的一个全人的TNF-α单克隆抗体，其可特异性地与TNF结合并阻断其与p55和p75细胞表面TNF受体的相互作用。但adalimumab单抗在宿主体内免疫原性较强，高达26％以上的患者使用该单抗会发生免疫反应，这给临床患者的使用带来了风险和不便，同时药物半衰期也相对较短，使用的过程中也需要增大药物剂量来弥补该方面的缺陷。基于此，若能够在不影响抗体亲和力和特异性的前提下，通过将抗体中高免疫原性的非抗原结合位点换成低免疫原性的同源序列来降低抗体药的免疫原性，则一方面可使单抗的安全性得到提高；另一方面可增加药物半衰期，在减少其用剂量同时也能起到提高疗效的作用。目前未见低免疫原性TNF-α的抗体的报道。
发明内容
本发明的第一个目的在于提供一种低免疫原性的全人源抗TNF-α的单克隆抗体。
本发明的另一个目的在于提供一种制备低免疫原性全人源抗TNF-α的 单克隆抗体的方法。
本发明利用商业化的DNAStarTM软件对阿达木单抗(购自美国雅培公司)的原始序列进行评价，结果显示，阿达木免疫原性系数为16。利用上述软件对抗体的可变区里面的非抗原结合片段(FR)进行免疫原性评价，找到免疫原性强的相关序列。随后，查找人抗体基因库中所有涉及轻重链的FR区段，通过比较后选择出一些免疫原性相对较低的区段。将区段中相应变动的部分分别得同阿达木单抗中对应的区段进行替换，随后用Pymol蛋白分子模拟软件进行3D建模，选择保持了原有的抗原结合位点的序列进行基因合成，测序，选择测序正确的序列。
本发明的低免疫原性抗TNF-α全人源单克隆抗体，即是在原始阿达木序列的轻链可变区和重链可变区的FR区进行氨基酸改造，降低其免疫原性，所述原始阿达木序列的轻链可变区和重链可变区的氨基酸序列分别如SEQ ID NO.23、24所示。
进一步地，本发明提供的低免疫原性抗TNF-α全人源单克隆抗体，其轻链可变区的氨基酸序列为SEQ ID NO.11～15或SEQ ID NO.23任一所示，其重链可变区的氨基酸序列为SEQ ID NO.16～20任一所示。
更进一步地，本发明提供的低免疫原性抗TNF-α全人源单克隆抗体，其轻链可变区的氨基酸序列如SEQ ID NO.13所示，其重链可变区的氨基酸序列如SEQ ID NO.19所示。编码其的核苷酸序列分别如SEQ ID NO.3和SEQ ID NO.9所示。
本发明提供的全人源的抗人TNF-α单克隆抗体，其轻链可变区具有L1至L5(SEQ ID NO1～5所示)这五条碱基序列所示的任一个轻链可变区序列，重链可变区具有h1至h5(SEQ ID NO.6～10所示)这五条碱基序列所示的任一个重链可变区序列。
具备上述轻链可变区和重链可变区的组合中，本发明共筛选得到10个单抗序列，它们既保持了原有抗原结合位点、又与人TNF-α结合的亲和力和原始adalimumab抗体相近，可特异性地阻断TNF-α与细胞表面TNF受体p55和p75的结合。
这10个单克隆抗体都具有相同的CDR区域且具有同样的功能，但对这10个抗体FR区域中某些氨基酸进行了不同的调整，这可使它们的免疫原性得到有效的降低。
本发明提供上述10种单抗的轻链可变区和重链可变区的编码基因。
本发明得到的10个低免疫原性的全人源抗TNF-α的单克隆抗体，分别为L3h2、L3h4、L5h2、L4h1、L4h2、L4h4、L1h3、L2h1、L0h4、L2h5。
其中L3h2单抗的轻链的核苷酸序列分别如SEQ ID NO.3所述，重链的核苷酸序列分别如SEQ ID NO.7所述，其轻链的氨基酸序列分别如SEQ ID NO.13所述，重链的氨基酸序列分别如SEQ ID NO.17所述。
其中L3h4单抗的轻链的核苷酸序列分别如SEQ ID NO.3所述，重链的核苷酸序列分别如SEQ ID NO.9所述，其轻链的氨基酸序列分别如SEQ ID NO.13所述，重链的氨基酸序列分别如SEQ ID NO.19所述。
其中L5h2单抗的轻链的核苷酸序列分别如SEQ ID NO.5所述，重链的核苷酸序列分别如SEQ ID NO.7所述，其轻链的氨基酸序列分别如SEQ ID NO.15所述，重链的氨基酸序列分别如SEQ ID NO.17所述。
其中L4h1单抗的轻链的核苷酸序列分别如SEQ ID NO.4所述，重链的核苷酸序列分别如SEQ ID NO.6所述，其轻链的氨基酸序列分别如SEQ ID NO.14所述，重链的氨基酸序列分别如SEQ ID NO.16所述。
其中L4h2单抗的轻链的核苷酸序列分别如SEQ ID NO.4所述，重链的核苷酸序列分别如SEQ ID NO.7所述，其轻链的氨基酸序列分别如SEQ ID NO.14所述，重链的氨基酸序列分别如SEQ ID NO.17所述。
其中L4h4单抗的轻链的核苷酸序列分别如SEQ ID NO.4所述，重链的核苷酸序列分别如SEQ ID NO.9所述，其轻链的氨基酸序列分别如SEQ ID NO.14所述，重链的氨基酸序列分别如SEQ ID NO.19所述。
其中L1h3单抗的轻链的核苷酸序列分别如SEQ ID NO.1所述，重链的核苷酸序列分别如SEQ ID NO.8所述，其轻链的氨基酸序列分别如SEQ ID NO.11所述，重链的氨基酸序列分别如SEQ ID NO.18所述。
其中L2h1单抗的轻链的核苷酸序列分别如SEQ ID NO.2所述，重链的 核苷酸序列分别如SEQ ID NO.6所述，其轻链的氨基酸序列分别如SEQ ID NO.12所述，重链的氨基酸序列分别如SEQ ID NO.16所述。
其中L2h5单抗的轻链的核苷酸序列分别如SEQ ID NO.2所述，重链的核苷酸序列分别如SEQ ID NO.10所述，其轻链的氨基酸序列分别如SEQ ID NO.12所述，重链的氨基酸序列分别如SEQ ID NO.20所述。
其中L0h4单抗的轻链的核苷酸序列分别如SEQ ID NO.21所述，重链的核苷酸序列分别如SEQ ID NO.9所述，其轻链的氨基酸序列分别如SEQ ID NO.23所述，重链的氨基酸序列分别如SEQ ID NO.19所述。
本发明提供了含有上述10个中任一个低免疫原性的全人源抗TNF-α的单克隆抗体轻链可变区和重链可变区基因的表达载体。含有所述表达载体的宿主菌、宿主细胞或表达盒也在本发明的保护范围内。
本发明提供上述10个低免疫原性的全人源抗TNF-α的单克隆抗体在制备治疗自身免疫性疾病药物中的应用。
所述的疾病为风湿性关节炎，红斑狼疮。
本发明提供了上述全人源抗TNF-α单克隆抗体在制备以TNF-α为靶标的疾病治疗药物中的应用。
所述的药物为抗肿瘤药、抗炎药物或治疗自身免疫性疾病的药物。
本发明提供含有上述全人源抗TNF-α单克隆抗体的药物或检测试剂。
本发明提供了制备上述全人源抗TNF-α单克隆抗体的方法，包括以下步骤：(1)对NCBI数据库里的人源抗体的FR序列进行分析(详见图2、3)，根据上述结果同时利用商业化的DNAstar软件对抗体的可变区里面的4个非抗原结合片段(FR)进行免疫原性评价，找到免疫原性强的片段。然后通过对已知的人抗体基因库里面找出所有重链的FR3段，挑选出一些免疫原性低的相应区段。将阿达木单抗上相应的重链及轻链区段进行替换，随后用Pymol蛋白分子模拟软件进行3D建模，结果发现大部分替换排列组合导致抗体的抗原结合位点构象变动很大，但是也有部分基本保持了原有的抗原结合位点；
(2)根据以上信息，本发明对相应修改后的序列进行全基因合成，对 合成的基因进行测序同时选择测序正确的序列进行下一步操作，将轻链可变区设计酶切位点为Kpn I+BamH I，重链可变区设计酶切位点为KpnI+AgeI，分别与表达载体pJH16载体连接，同时转化大肠杆菌DH5α，得到重链、轻链嵌合抗体表达载体，结果详见图1。同时对构建的抗体进行测序和序列对比；
(3)对上述表达的载体进行质粒大提工作，选取Qiagen的无内毒素质粒大提试剂盒(详见该质粒试剂盒说明书)；
(4)对选取的质粒进行优化组合，同时利用293F细胞进行瞬时转染表达，对表达的抗体进行亲和力和EC50检测(详见图4)根据检测结果来选定哪些组合进行稳定转染；
(5)根据上述检测结果，本发明选取相应的组合利用电转染方式构建稳定株，同时利用MTX进行抗体表达程度的筛选，对于加压后的稳定株进行单克隆筛选，最终挑选出抗体产量较高的稳定株，用于后续实验所用；
(6)本发明生产的单克隆抗体在小鼠体内的免疫反应比adalimumab低，同时半衰期明显延长。
本发明提供的全人源抗TNF-α抗体和adalimumab针对的是TNF-α相同位点，但其免疫原性和抗体构象同adalimumab不同，同adalimumab相比其药物半衰期延长，有望成为非常理想的生物靶向治疗抗体。本发明通过对adalimumab单抗进行免疫原性改造，使抗体中部分氨基酸序列改变，将显著降低该抗体药在病人体内产生免疫原性的风险。本发明实施例显示本发明的改进型adalimumab抗体与TNF-α结合的亲和力和原始adalimumab相近，并延长抗体药的半衰期，提高疗效。
附图说明：
图1为质粒及合成基因酶切结果；其中a图为pJH16质粒双酶切结果(kpn Ⅰ和Age Ⅰ)，1：质粒双酶切结果。b图为pJH16质粒双酶切结果(kpn Ⅰ和BamH Ⅰ)，1：质粒双酶切结果。c图为阿达木原始重链(kpn Ⅰ和Age Ⅰ)及轻链(Kpn I+BamH I)基因双酶切结果，1：重链双酶切结果，2： 轻链双酶切结果；其中M为DL 15000marker。
图2人源化轻链氨基酸序列BLAST比对结果；其中，图2a、图2b、图2c三个图均为该BLAST比对结果。
图3人源化重链氨基酸序列BLAST比对结果；其中，图3a、图3b、图3c三个图均为该BLAST比对结果。
图4改进型阿达木亲和力EC50。
图5改进型阿达木抑制TNF-α诱导L929细胞毒性实验。其中，图5a代表TNF-α诱导L929细胞毒性实验，作为实验的阳性对照；图5b代表改进型阿达木抗体抑制TNF-α诱导L929细胞毒性。
图6改进型阿达木抗体药代动力学检测。
具体实施方式
以下实施实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1降低免疫原性阿达木序列的分析及设计
利用商业化DNAStarTM软件对阿达木原始序列进行评价，评价结果显示，阿达木免疫原性系数为16，针对此本发明需要进行下一步免疫原性的降低工作。
通过利用上述软件对抗体的可变区里面的非抗原结合片段(FR)进行免疫原性评价，找到免疫原性强的相关序列。随后，查找人抗体基因库中所有涉及轻重链的FR区段，通过比较后选择出一些免疫原性相对较低的区段。基于上述信息，将区段中相应变动的部分分别得同阿达木单抗中对应的区段进行替换，初步得到的全人源的抗人TNF-α单克隆抗体，其轻链可变区选自L1～L5、L0(SEQ ID NO.1～5、所示SEQ ID NO.21)任一个碱基 序列组成的轻链可变区序列，重链可变区选自h1～h5(SEQ ID NO.6～10)所示任一个碱基序列组成的重链可变区序列。
随后用Pymol蛋白分子模拟软件进行3D建模，结果发现大部分替换排列组合导致抗体的抗原结合位点构象变动很大，但是也有部分基本保持了原有的抗原结合位点；因此对保持原有抗原结合位点的修改后的所有序列进行全基因合成(委托金唯智公司合成)，对合成的基因进行测序(委托金唯智公司进行测序工作)，根据测序比对结果选择正确的序列。
实施例2阿达木单抗表达载体的构建
根据实施例1测序得到的改进型阿达木重链、轻链可变区碱基序列，设计轻链序列两侧的酶切位点为Kpn I+BamHI，设计重链序列两侧的酶切位点为Kpn I+AgeI，以上序列送交金唯智公司合成全基因序列，合成所用的连接载体为pUC57。以pJH16为表达载体，合成基因返回后，将它们分别与金唯智公司合成的序列进行酶切后(见图1)，16℃过夜连接。将酶切后得到的目的基因和表达载体(pJH16)进行琼脂糖凝胶电泳分离，随后切下目的条带并用Qiagen Gel Extraction Kit回收，按T4DNA连接体系连接过夜，然后转化大肠杆菌DH5α，对相应的菌株进行质粒提取和序列测定工作，测序结果表明二者序列完全一致，这表明抗体表达载体构建成功。
实施例3改进型阿达木单抗的瞬时表达与纯化
用实施例2构建的pJH16重链、轻链表达载体转染大肠杆菌DH5a。接种于100m1 LB培养基中，按照常规方法进行培养。收获培养物，用Qiagen公司的UltraPure质粒DNA纯合试剂盒抽提纯化质粒DNA。将上述纯化的质粒DNA采用Invitrogen公司的脂质体法试剂盒转染293F细胞，操作参照厂家的说明书进行。
首先对293F细胞进行不同轻、重链质粒组合的转染，组合见表1，共31组293F瞬时表达，需要从这31组中筛选出免疫原性降低的组合。培养3天后，取培养上清液，进行抗体表达量检测，结果如下表1所示：
表1：原始及改进型阿达木抗体表达量检测结果(ng/ml)
Figure PCTCN2015074528-appb-000001
Figure PCTCN2015074528-appb-000002
(注：表中组合是不同轻重链的组合，L0h1即指原始阿达木轻链可变区与本发明改进型阿达木重链可变区h1的组合，以此类推。Adh0l0是原始阿达木的组合)
对上述表达后的抗体分别进行预包TNF-α实验，从中筛选出较好的组别，实验结果如下：
加入预包有TNF-α的96孔板中，采用ELISA间接法，初步评价分泌抗体结合TNF-α的活性。上述31组的检测结果如下表2所示。NC为以抗体稀释液作为阴性对照。
表2：筛选的不同重链、轻链组合的抗体活性评价结果
Figure PCTCN2015074528-appb-000003
实施例4改进型阿达木单抗的稳定表达与纯化
根据上述检测结果，选取质检结果较优的10个组合进行稳定转染。
采用电转染方法转染CHO(购自美国英俊公司)细胞，并利用选择性培养基Opti-CHO(美国英俊-opti-cho medium)进行MTX加压筛选(MTX购自sigma公司)，分别以50、100、200、400、800nM这五个梯度进行加压，对于每轮加压后的细胞进行7d滴度测定，测定方法采用ELISA-双抗夹心法确定每步加压后工程细胞株的抗体产量。该过程完成后，利用有限稀释法进行单克隆筛选，该方法主要是通过对细胞进行稀释铺板(96孔板)， 在37℃5％CO2条件下培养大约14天后取50微升进行抗体产量初筛(采用ELISA-双抗夹心发测定)对于筛选结果较好的进行克隆挑取并扩大培养。
部分结果如表3：结果显示，不同轻重链的组合均得到表达，工程细胞株产生了相应的抗体(ELISA评价结果数据)。
表3：不同重链、轻链组合的抗体表达浓度评价
Figure PCTCN2015074528-appb-000004
抗体纯化部分工作利用GE公司的Protein A进行纯化，选取200nM加压组进行抗体稳定株生产工作，利用Protein A亲和层析柱对上述11个稳定细胞系的培养上清直接分离纯化进而得到本发明的人源单克隆抗体(注：其中10个为改进型，1个为原始adalimumab)。实验过程详见GE公司说明书，纯化后产品利用紫外分光光度计进行定量检测，计算公式如下：
浓度计算：将收集的洗脱峰读OD280后，计算浓度。抗体浓度＝OD280/1.4。抗体纯化下来后进行SDS-PAGE电泳验证。
实施例5改进型阿达木单抗的生物活性测定
1、亲和力评价
本部分利用ELISA间接法测抗体EC50评价抗体亲和力。
实验方法如下：用PBS将TNF-a稀释抗原到0.3ug/ml；将稀释好的抗原按100ul/孔加到96孔板中，加盖，4℃过夜；甩去孔内液体，用PBS洗三次，200ul/孔，拍干；用5％牛乳-PBS 200ul/孔封闭1h，每15min轻拍； 甩去孔内液体，用PBS洗一次，200ul/孔，拍干；分梯度加入纯化抗体(0-10ug/ml)，抗体名称见表4，5％牛乳-PBS稀释，100ul/孔，孵育1h，每15min轻拍；甩去孔内液体，用PBS洗三次，200ul/孔，拍干；加二抗，5％牛乳-PBS稀释，100ul/孔，孵育1h，每15min轻拍；预热TMB底物于室温，同时开启酶标仪预热；PBS洗5次，250ul/well，前三次5min，后两次10min，拍干；TMB底物A B各加50ul/孔，室温显色20min；使用扫描仪将图片扫描记录，加入终止液50ul/孔，使用酶标仪读取OD450。实验结果见图4。实验数据见下表4。
表4：优选的改进型阿达木轻重链可变区组合的EC50结果
组合名称 l0h4 l2h1 l3h2 l3h4 l4h1 l4h2 l4h4 adh0l0
EC50(nM) 0.37 0.43 0.40 0.34 0.47 0.60 0.69 0.49
以上结果表明：改进型阿达木亲和力与原始阿达木亲和力基本一致，有的甚至比原始阿达木要高。
2、特异性评价
该过程验证所表达的抗体是否有针对rhTNFα具有特异性，利用不同的因子包板并采用间接法测定。
具体实验过程如下：用PBS将rhTNFα、rhTNFβ、rIFNγ(重组人干扰素γ)，IL-1α、IL-1β、IL-2、IL-4和IL-8，稀释抗原到1ug/ml；将稀释好的抗原100ul/孔加到96孔板中，并加盖，4℃过夜；甩去孔内液体，用PBS洗三次，200ul/孔，手动拍干；用5％牛乳200ul/孔封闭，封闭1h，每15min轻拍酶标板边以促进反应；甩去孔内液体，用PBS洗一次，200ul/孔，手动拍干；加不同轻链重链组合的抗体，5％牛乳稀释，100ul/孔，孵育1h，每15min轻拍酶标板边以促进反应；甩去孔内液体，用PBS洗三次，200ul/孔，手动拍干；加入羊抗人二抗，5％牛乳稀释，100ul/孔；孵育1h，每15min轻拍酶标板边以促进反应；预热TMB底物于室温，同时开启酶标仪预热；PBS洗5次，250ul/孔，前三次5min，后两次10min，手动拍干；TMB底物A B各加50ul/孔，室温显色20min；使用扫描仪将图片扫描记录，加入终止液50ul/孔，使用酶标仪读取OD450，存档。实验结果如表5。
表5：全人源阿达木改进型单抗特异性检测
Figure PCTCN2015074528-appb-000005
以上结果表明：改进型阿达木单抗针对rhTNFα具有特异性
3、细胞毒作用实验
本部分使用CCK-8试剂盒进行相应的实验，本实验主要利用小鼠成纤维细胞株L929进行细胞凋亡的检测。具体操作步骤如下：
在96孔板中加入100ul三倍梯度稀释的改进型阿达木单抗(RPMI-1640培养基含10％FBS稀释)(改进型阿达木单抗加入浓度依照FDA中提及阿达木此实验IC50分别上下3-5个梯度浓度)，随后加入50ul终浓度为500pg/ml的rhTNF-α(RPMI-1640培养基含10％FBS稀释)，室温孵育30min。向每孔加入50ul L929细胞5×104/孔,包含终浓度为1ug/ml的放线菌素-D。37℃培养箱孵育过夜培养(18-24h)。对照包括阴性对照和阳性对照，阴性对照只加入RPMI-1640及细胞，阳性对照只加入rhTNFα及细胞，rhTNFα浓度从2ng/ml到8.2pg/ml梯度。向每孔加入20μL CCK8溶液(注意不要在孔中生成气泡，它们会影响OD值的读数)。将培养板 在37℃培养箱内孵育1-4h。用酶标仪测定在470nm处的吸光度。若暂时不测定OD值，可以向每孔中加入10μL 0.1M的HCL溶液或者1％w/v SDS溶液，并遮盖培养板避光保存在室温条件下。24小时内测定，吸光度不会发生变化。
计算抗体在不同浓度下的细胞杀伤率和IC50(阿达木的细胞毒性IC50值：1.25±0.01e-10M)
计算公式：改进阿达木处理细胞—TNF-α处理细胞/TNF-α细胞×100％
结果显示，本发明的改进型阿达木单抗与原始阿达木单抗相比，抑制TNF-α诱导的细胞毒作用相当(图5b)。
实施例6改进型阿达木单抗免疫原性评价
1.小鼠免疫实验
1)基础免疫：将抗原与福氏完全佐剂等体积混合并充分乳化，分点皮下注射，每只Balb/c小鼠每次注射量为100μg。
2)加强免疫：加强免疫采用抗原与福氏不完全佐剂的乳化液。
上述实验完成后进行ELISA检测试验，结果见表6。
表6：小鼠免疫原性评价实验
Figure PCTCN2015074528-appb-000006
上述结果表明，本发明的10个改进型阿达木单抗同原始阿达木单抗相比，免疫原性得到了有效的降低。
2.抗体生物分布和药代试验
分别对原始阿达木及改进型阿达木进行抗体生物分布实验及药代试验，基本过程如下：
抗体生物分布：选取16只荷瘤裸鼠随机分为4组，每组4只。每只由尾静脉注射563.5kBq(100μL,2.5μg)125I-抗体，并于注射后2、4、8和24h，按组将实验裸鼠断颈处死，取血液及主要脏器和组织，称重并量放射性计数，计算各器官或组织的百分注射剂量率(％ID/g)。
表7：改进型阿达木抗体分布检测
Figure PCTCN2015074528-appb-000007
药代分布试验：取5只BALB/c小白鼠，每只经尾静脉注射100μL125I-抗体(370kBq,2μg)，分别于注射后不同时间点(5、12、30min,1、2、4、8、11、22、34、48、72h)眼眶取血，称重并用γ计数仪测量其放射性计数，计算每克血液百分注射剂量率(％ID/g)。采用Grap h Prism软件进行分析，计算药代动力学参数。结果发现本发明的10个改进型阿达木单抗比原始阿达木单抗的半衰期长(图6)。
工业实用性
本发明提供的低免疫原性的抗TNF-α全人源单抗与人TNF-α结合的亲和力和原始adalimumab抗体相近，可特异性地阻断TNF-α与细胞表面TNF受体p55和p75的结合。本发明的低免疫原性的抗TNF-α全人源单抗可用于制备针对TNF-α靶标的抗肿瘤及其他疾病如炎症及自身免疫性疾病的预防和治疗性抗体药物，该抗体药有望降低其在病人体内产生免疫原性的风险，延长抗体药的半衰期，提高疗效，具有优异的临床应用价值。
Figure PCTCN2015074528-appb-000008
Figure PCTCN2015074528-appb-000009
Figure PCTCN2015074528-appb-000010
Figure PCTCN2015074528-appb-000011
Figure PCTCN2015074528-appb-000012
Figure PCTCN2015074528-appb-000013
Figure PCTCN2015074528-appb-000014
Figure PCTCN2015074528-appb-000015
Figure PCTCN2015074528-appb-000016
Figure PCTCN2015074528-appb-000017
Figure PCTCN2015074528-appb-000018
Figure PCTCN2015074528-appb-000019
Figure PCTCN2015074528-appb-000020
Figure PCTCN2015074528-appb-000021

Claims (10)

  1. 一种低免疫原性抗TNF-α全人源单克隆抗体，其特征在于，在原始阿达木序列的轻链可变区和重链可变区的FR区进行氨基酸改造，降低其免疫原性，所述原始阿达木序列的轻链可变区和重链可变区的氨基酸序列分别如SEQ ID NO.23、24所示。
  2. 如权利要求1所述的低免疫原性抗TNF-α全人源单克隆抗体，其特征在于，其轻链可变区的氨基酸序列为SEQ ID NO.11～15或SEQ ID NO.23任一所示，其重链可变区的氨基酸序列为SEQ ID NO.16～20任一所示。
  3. 如权利要求1所述的单克隆抗体，其特征在于，其轻链可变区的氨基酸序列如SEQ ID NO.13所示，其重链可变区的氨基酸序列如SEQ ID NO.19所示。
  4. 编码权利要求1～3任一所述单克隆抗体的基因。
  5. 含有权利要求4所述的基因的表达载体。
  6. 权利要求4所述的基因或权利要求5所述的表达载体在制备治疗以人TNF-α为靶标的疾病药物中的应用。
  7. 权利要求1～3任一所述单克隆抗体在制备治疗以人TNF-α为靶标的疾病药物中的应用。
  8. 权利要求1～3任一所述单克隆抗体在制备治疗自身免疫性疾病药物中的应用。
  9. 如权利要求8所述的应用，其特征在于，所述的疾病为风湿性关节炎、红斑狼疮。
  10. 含有权利要求1～3任一所述单克隆抗体的药物或检测试剂。
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