mVEGF表位疫苗在小鼠移植瘤的应用

发布时间：2019-06-02 04:42

【摘要】：肿瘤的生长离不开营养的供给，因此，肿瘤新生血管的形成对于肿瘤的生长具有重要意义。在众多血管生成有关的细胞因子中，VEGF是最重要的调节因子，很多肿瘤的生长过程都伴随着VEGF的高表达，许多研究表明，抑制VEGF的生物学活性就能抑制肿瘤的生长。目前，用VEGF抗体治疗肿瘤取得了一定的临床效果，但仍存在许多不足。临床使用的VEGF抗体为人源化抗体，虽然人源化程度较高，但仍存在异源性的问题。由于临床上用抗体治疗肿瘤需要反复多次用药，因此，抗体本身的异源性问题不容忽视。另外，抗体临床用量较大，其生产成本较高，导致抗体药物的价格较高，也一定程度上限制了其使用范围。用外源VEGF抗体治疗肿瘤是一种被动免疫治疗。如果能够采用主动免疫治疗，则可以避免VEGF抗体治疗的一些不足，如用药次数多、存在异源性等问题。但是VEGF是机体自身产生的蛋白质，有其正常的生理功能，机体对正常的自身蛋白存在免疫耐受，用自身VEGF蛋白难以诱导机体产生针对自身VEGF的抗体。本文的目的就是要设计一种mVEGF疫苗，绕开或打破机体的免疫耐受，让机体能够产生识别mVEGF的中和性抗体，以抑制肿瘤新生血管的生成，最终实现抑制肿瘤生长的目的。为了实现上述目的，我们确定了mVEGF疫苗设计的基本策略，即以mVEGF空间结构为依据，寻找mVEGF可能的抗原表位，将这些可能的抗原表位展示在一种简单的蛋白质骨架之上，形成一种新蛋白，用这种新蛋白作为可能的mVEGF疫苗。首先，我们选择了抗体重链可变区作为抗原表位展示的骨架蛋白，由于抗体的抗原结合部位是由抗体的轻链和重链可变区共同组成，单独的重链抗体可变区存在稳定性差的缺点，经过分析，我们将抗体重链可变区的部分氨基酸进行了突变，突变后的重链可变区蛋白可以很容易地实现原核表达，并可复性为稳定的蛋白质。由于抗体的重链可变区的CDR3区的是高度可变的，能够容忍不同长度序列的插入或置换，因此，CDR3区被我们选为抗原表位展示部位。根据人VEGF165的空间结构，我们分析了小鼠VEGF164的可能的抗原表位，其中表位1（EYPDEIEYIFKP）、表位2（KSHEVIKFMDV）和表位3（IMRIKPHQSQH）有可能是mVEGF的中和性抗原表位。我们用表位2的氨基酸序列替换抗体重链可变区的CDR3区氨基酸序列，形成一种新的蛋白质，命名为mFV2。将此序列转换为其基因编码序列，全基因合成其编码序列，并克隆到pET-24a表达载体上，构建了表位2表达载体。将该载体转化BL21（DE3）宿主菌，，通过IPTG诱导实现了mFV2的高表达，表达的mFV2蛋白主要以包涵体形式存在，包涵体经过洗涤、纯化、裂解后，用SephacrylS-100凝胶过滤柱进行了纯化，获得了高纯度的mFV2蛋白，通过稀释法成功复性了mFV2蛋白。通过重叠PCR法将表位1和表位3编码基因成功插入到抗体可变区载体蛋白的CDR3区中，同样实现了mFV1和mFV3的表达、纯化和复性。在获得了三种小鼠表位疫苗蛋白mFV1、mFV2和mFV3后，我们首先验证了三种疫苗蛋白的免疫效果。用三种疫苗蛋白分别免疫雌性Balb/c小鼠，免疫5周后小鼠眼眶取血，获得三种免疫血清，命名为pAb1、pAb2和pAb3。ELISA检测表明，三种免疫血清的滴度均达到1：104，并且能够特异性识别VEGF164，而不与无关蛋白反应，这表明,这三种疫苗蛋白的确能够诱导机体产生特异性识别VEGF164的抗体。WesternBlot检测显示，免疫血清pAb1和pAb2能够识别VEGF164，但pFV3则不能识别VEGF164，很可能表位3是一种空间表位，在WesternBlot中该表位不能保持其空间结构。另外，通过免疫荧光实验证明，三种免疫血清都能识别B16小鼠黑色素瘤细胞中表达的VEGF164，B16细胞表达的VEGF164主要分布在胞浆中。为了验证三种免疫血清是否具有VEGF164中和活性，我们分析了三种免疫血清对人脐静脉内皮细胞（HUVEC）增殖、迁移和成管能力的影响。实验结果表明，VEGF164能够促进HUVEC的增殖、迁移及成管，而pAb1、pAb2、pAb3均表现出了一定的mVEGF抑制活性，其中pAb3表现出了理想的VEGF164抑制活性。上述体外实验结果证明，三种免疫血清的确具有VEGF164中和活性，能够抑制VEGF164的生物学功能。在此基础上，我们开展了mVEGF疫苗治疗肿瘤的实验研究。首先，我们采用预防接种的方式观察三种mVEGF疫苗是否具有抑制H22 实体瘤生长的作用。实验选用6-8周的雌性BALB/c小鼠，分为四组，分别是模型组、mFV1组、mFV2组和mFV3组。mFV1组、mFV2组和mFV3组分别用mFV1蛋白、mFV2蛋白和mFV3蛋白免疫三次，第5周通过ELISA检测免疫效果，并开始接种H22肿瘤细胞，待肿瘤生长到可用手摸到时开始记录肿瘤生长状态，并最终处死小鼠，分离肿瘤，称量肿瘤大小。实验结果表明，三种mVEGF疫苗均表现出了一定的抑制H22肿瘤生长的能力，其中，mFV3疫苗的效果最理想，抑瘤率达到83.8%，mFV2次之，mFV1最差。为了更直观地了解mVEGF疫苗的抑瘤机制，将H22肿瘤模型实验中各组的肿瘤组织进行了免疫组化检测，结果显示，疫苗治疗组微血管密度比模型组低，其中mFV3免疫组肿瘤的微血管密度减少最为明显，这就是为什么在三种疫苗蛋白中mFV3抑瘤作用最好的原因。同样地，利用S180肿瘤模型验证了三种mVEGF疫苗的抑瘤效果。在S180肿瘤模型上，三种疫苗的抑瘤表现与在H22肿瘤模型上的表现一致，也是mFV3疫苗的效果最理想，抑瘤率为80.3%，mFV2次之，mFV1最差。为了模拟临床上疫苗使用的实际情况，我们又开展了治疗性接种mVEGF疫苗对肿瘤生长影响及与化疗药物共用的效果观察。实验仍选用6-8周的雌性BALB/c小鼠，分为四组，分别是模型组、mFV3治疗组、顺铂治疗组和mFV3+顺铂组。实验中接种H22肿瘤细胞与给药同时进行，模型组仅注射生理盐水，顺铂组注射顺铂2mg/kg，每周一次，连续用药2次，mFV3+顺铂组为顺铂和mFV3联合治疗，小鼠接种肿瘤细胞后，注射顺铂（2mg/kg）并同时进行mFV3免疫，顺铂每周一次，连续用药2次，第14天进行mFV3第二次免疫，于第28天处死小鼠，并剥离肿瘤组织，测量肿瘤大小。实验结果为，单纯mFV3免疫组肿瘤抑制率为27.3%,单纯顺铂治疗组肿瘤抑制率为35.7%，而mFV3免疫+顺铂治疗组的肿瘤抑制率达到了66.2%。可见，mVEGF疫苗治疗和化疗之间并不冲突，二者有较好的协同效应，可联合用于肿瘤的治疗。综上所述，本文建立了一种基于抗体可变区的抗原表位展示系统，利用该系统构建了三种mVEGF疫苗，它们能够有效诱导小鼠产生VEGF164中和抗体，最终抑制小鼠实体瘤的生长。
[Abstract]:The growth of the tumor is essential for the growth of the tumor, so the formation of the tumor-based new blood vessel is of great significance to the growth of the tumor. VEGF is the most important regulatory factor in many angiogenesis-related cytokines, and the growth of many tumors is associated with the high expression of VEGF, and many studies have shown that the inhibition of the biological activity of VEGF can inhibit the growth of the tumor. At present, the treatment of the tumor with the VEGF antibody has achieved a certain clinical effect, but there are still many problems that do not The VEGF antibody used in clinical use is a humanized antibody, although the humanizing degree is high, there is still a heterogenous question. in that clinical use of the antibody for the treatment of the tumor, repeated use of the medicament is required, therefore, the problem of the heterogenous problem of the antibody itself is not allowed to be In addition, the clinical dosage of the antibody is large, the production cost is high, the price of the antibody drug is high, and the use of the antibody is limited to a certain extent. Perimeter. The treatment of a tumor with an exogenous VEGF antibody is a passive if active immunotherapy can be used, some deficiency of VEGF antibody treatment can be avoided, However, VEGF is the protein produced by the body itself, has its normal physiological function, and the body is immune tolerance to the normal self-protein, and it is difficult to induce the body to produce the self-VEGF by using the self-VEGF protein. The purpose of the present invention is to design a mVEGF vaccine, to bypass or to break the immune tolerance of the body, to enable the body to generate neutralizing antibodies to recognize the mVEGF, to inhibit the generation of the tumor new blood vessels, and finally to realize the inhibition of the tumor. In order to achieve the above objects, we have determined the basic strategy of the design of mVEGF vaccine, i. e., based on the space structure of mVEGF, to find the potential epitope of mVEGF, to show these potential epitopes on a simple protein skeleton, to form a new protein that is possible with this new protein mVEGF vaccine. First, we selected the antibody heavy chain variable region as the skeleton protein shown by the epitope of the antigen, because the antigen binding site of the antibody is composed of the light chain and the heavy chain variable region of the antibody, the single heavy chain antibody variable region has stability The disadvantage of the difference is that, after the analysis, we have mutated some of the amino acids in the variable region of the antibody heavy chain, and the mutant heavy chain variable region protein can easily realize the prokaryotic expression and can Renaturation is a stable protein. Since the CDR3 region of the heavy chain variable region of the antibody is highly variable, insertion or displacement of the different length sequences can be tolerated, so that the CDR3 region is selected by us The potential epitope of the mouse VEGF164 is analyzed according to the spatial structure of the human VEGF165, wherein the epitope 1 (EYPDEIEYIFKP), the epitope 2 (KSHEVIKFMDV) and the epitope 3 (IMRIKPHQSQH) may be mV. The neutralizing antigen epitope of the EGF is formed by replacing the amino acid sequence of the CDR3 region of the heavy chain variable region of the antibody with the amino acid sequence of the epitope 2, The novel protein is named mFV2. The sequence is converted into its gene coding sequence, the whole gene is synthesized with its coding sequence and cloned into pET-24a expression carrier. and the expression vector of the epitope 2 is constructed on the body, the vector is transformed into the BL21 (DE3) host strain, the high expression of the mFV2 is realized through the induction of IPTG, the expressed mFV2 protein is mainly in the form of an inclusion body, the inclusion body is washed, purified and cracked, and purified by a Sephacryl S-100 gel filtration column, high-purity mFV2 protein is obtained, The mFV2 protein is successfully renatured by the release method. The epitope 1 and the epitope 3 encoding gene are successfully inserted into the CDR3 region of the antibody variable region vector protein by the overlapping PCR method, and the mFV is also realized. Expression, purification and renaturation of 1 and mFV3. After three mouse epitope vaccine proteins mFV1, mFV2 and mFV3 were obtained, I The three kinds of vaccine proteins were used to immunize the female Balb/ c mice, and the mice were immunized for 5 weeks to obtain three kinds of immune serum, named pAb1, pAb2 and pAb3. The ELISA test showed that the titer of the three kinds of immune serum was 1:104, and it was able to specifically recognize the VEGF. 164, without reacting with extraneous proteins, suggesting that these three vaccine proteins are indeed capable of inducing the body The antibodies that specifically identify the VEGF164 are produced. The Western Blot test shows that the immune serum pAb1 and pAb2 are able to recognize the VEGF164, but pFV3 does not recognize the VEGF164, and it is likely that the epitope 3 is a space epitope, in Western The epitope in Blot cannot maintain its spatial structure. In addition, by immunofluorescence experiments, all three kinds of immune serum can recognize the expression of VEGF164 and B16 cell in B16 mouse melanoma cells. The amount of VEGF164 is mainly distributed in the cytoplasm. In order to verify that the three kinds of immune serum have VEG In F164 and activity, we analyzed three kinds of immune serum on human umbilical vein endothelial cells ( The results showed that VEGF164 could promote the proliferation, migration and tube formation of HUVEC, while pAb1, pAb2, pAb3 showed a certain mVEGF inhibitory activity, of which pAb 3. The results of the above-mentioned in vitro test show that the three kinds of immunity Serum does have a neutralizing activity of VEGF164 Sex, can inhibit the biological function of VEGF164. On this basis, We carried out an experimental study of the treatment of tumors with the mVEGF vaccine. First of all, we use a prophylactic vaccination. The three types of mVE were observed. Whether the GF vaccine has the effect of inhibiting the growth of H22 solid tumor. In the experiment,6-8 weeks of female BALB/ c mice were selected and divided into four groups. In the model group, mFV1 group, mFV2 group and mFV3 group, mFV1 group, mFV2 group and mFV3 group were immunized three times with mFV1 protein, mFV2 protein and mFV3 protein, and the immune effect was detected by ELISA at week 5, and H22 tumor cells were inoculated, and when the tumor was grown to the available hand, start to record the tumor The results showed that the three mVEGF vaccines showed a certain ability to inhibit the growth of H22 tumor, and the effect of mFV3 vaccine was the most ideal. The tumor rate was 83.8%, mFV2 was the second, and mFV1 was the worst. In order to understand the mV more intuitively, The anti-tumor mechanism of the EGF vaccine was carried out by the immunohistochemical staining of the tumor tissues in the H22 tumor model. The results showed that the microvessel density ratio in the vaccine treatment group was higher than that of the model group. The decrease of the microvessel density in the mFV3 immune group is the most obvious, which is It is the best reason why mFV3 is the best in the three vaccine proteins. Similarly, The tumor-inhibiting effect of three kinds of mVEGF vaccines is verified by using the S180 tumor model. In the S180 tumor model, the tumor-inhibiting performance of the three vaccines is consistent with the expression on the H22 tumor model, and also the effect of the mFV3 vaccine The most ideal, the tumor-inhibiting rate is 80.3%, mFV2 is the second, and mFV1 is the worst. In order to simulate the actual situation of the clinical vaccine, we have also carried out the treatment The effect of the mVEGF vaccine on the growth of the tumor and the effect of the administration of the mVEGF vaccine on the growth of the tumor were observed. The 6-8 weeks of female BALB/ c mice were selected and divided into four groups. The model group, the mFV3 treatment group, the cisplatin treatment group, and the mFV3 + cisplatin group were respectively inoculated. In the experiment, the H22 tumor cells were inoculated with the administration, and the model group was injected with normal saline only, and the cisplatin group was injected with cisplatin for 2 mg/ kg once a week. In combination with cisplatin and mFV3, the FV3 + cisplatin group was treated with cisplatin and mFV3. After the mice were inoculated with tumor cells, cisplatin (2 mg/ kg) was injected and mFV3 immunization was performed at the same time. Cisplatin was administered once a week for 2 times, and mFV3 was performed on day 14. After the second immunization, the mice were sacrificed on day 28, and the tumor tissues were removed and the size of the tumor was measured. The experimental results were as follows: the tumor inhibition rate of the pure mFV3 group was 27.3%, and the tumor inhibition rate of the pure cisplatin group was 35.7%. %, and the tumor inhibition rate of mFV3 + cisplatin-treated group reached 66.2%. In conclusion, an antigen epitope display system based on an antibody variable region is established, and three mVEGF vaccines are constructed by using the system,
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【共引文献】