1、多發(fā)性骨髓瘤基因修飾瘤苗誘導(dǎo)體內(nèi)抗腫瘤反應(yīng)的實(shí)驗(yàn)研究    作者：任素萍，王立生，郭強(qiáng)，王華，賈向旭，徐娟，王恒湘，吳祖澤    【摘要】 本研究目的是評價NOD/SCID小鼠皮下移植瘤模型對多發(fā)性骨髓瘤基因修飾瘤苗引發(fā)體內(nèi)抗腫瘤反應(yīng)的效果。首先給NOD/SCID小鼠腹腔注射人外周血淋巴細(xì)胞以在其體內(nèi)重建人的免疫系統(tǒng)，然后皮下接種-射線滅活的基因修飾骨髓瘤細(xì)胞sko-007（表達(dá)綠色熒光蛋白或者p53、GM-CSF和B7-1基因），以PBS作為對照，最后植入活sko-007細(xì)胞進(jìn)行攻擊。結(jié)果發(fā)現(xiàn)，與對照組相比接種感

2、染腺病毒Ad-p53/GM-CSF/B7-1的sko-007 細(xì)胞可以明顯抑制移植瘤生長，病理分析顯示移植瘤纖維組織增生伴彌漫性壞死增多，血管增生顯著。免疫組織化學(xué)染色顯示瘤灶內(nèi)有人T淋巴細(xì)胞浸潤。結(jié)論： p53、GM-CSF和B7-1基因修飾的骨髓瘤細(xì)胞能夠誘導(dǎo)產(chǎn)生抗腫瘤免疫反應(yīng)，有可能用于人類多發(fā)性骨髓瘤的免疫治療。    【關(guān)鍵詞】 多發(fā)性骨髓瘤 Multiple myeloma (MM) remains an incurable malignancy despite advances in chemotherapy. Myeloablative

3、chemotherapy followed by allogeneic or autologous hematopoietic stem cell transplantation has increased the incidence of complete remission，but almost all patients achieving complete remission ultimately experience relapse1-3. Because these chemotherapies have only limited value，alternative strategi

4、es are needed to solve these problems. Immunotherapy may represent a means of maintaining complete remission.    Based on the fact that myeloma cells contain a multitude of tumor antigens that can effectively stimulate antitumor T cells，several investigators have repor- ted immun

5、otherapeutic approaches via inoculating mye- This project was supported in part by Chinese National Basic Research and Development Grants 973 (No. 2004CB518801 and No. 2002 CB713804)，Chinese High-Tech Program 863 (No. 2003AA216050) and Chinese National Science Foundation (No.30400189). Corresponding

6、 author: WU Chu-Tse (吳祖澤)，Academician of Chinese Academy of Sciences，Professor of Department of Experimental Hematology，Beijing Institute of Radiation Medicine. loma cell lysates，whole myeloma cells or genetically modified myeloma cell vaccines to augment the immunogenicity of myeloma cells. Numerou

7、s applications of genes encoding tumor suppressive proteins，cytokines and costimulatory molecules have been proposed in cancer (including MM) therapy4-7. Although the results of most preclinical investigations carried out in vitro and in mouse models were encouraging，the clinical evaluation is less

8、satisfactory8，9. Further studies found that rather than the absence of tumor-specific antigen，myeloma cells escape from immune surveillance mainly by down-regulating the expression of costimulatory molecules and inhibiting induction and maturation of dendritic cells (DCs)6，10-11. Hence a combination

9、 of immune-stimulating genes should be more efficient than any single gene. In previous study，we have de-monstrated that whole myeloma cell vaccination co-transferred with human wild-type p53，GM-CSF and B7-1 genes mediated by recombinant adenovirus Ad-p53/GM-CSF/B7-1 induces allologous and autologou

10、s specific anti-tumor cytotoxicity in vitro12. In this study we investigated whether the generation of Ad-p53/GM-CSF/B7-1-mediated immunity is protective against subsequent tumor challenge. NOD/SCID mice are the most immunodeficient of the SCID variants，and are the most supportive host for human ste

11、m cells13. Most SCID mouse myeloma models have used mouse or human MM cell lines. In 2 studies，fresh BMC from MM patient survived in conventional SCID mice，and in one group it was observed that human MM cell lines colonized in human fetal bone implanted subcutaneously in SCID mice14-16. However，all

12、the mice myeloma models could not reflect the interaction between human immune system and human myeloma cells.    HuPBL-NOD/SCID mouse model has been applied in several other tumors17-18. In this study，we intraperitoneally injected human PBL into NOD/SCID mice to establish human

13、immune system，innoculated the animals with genetically modified human myeloma cell vaccine，and then challenged subcutaneously the mice with live parental myeloma cells. The result showed that p53，GM-CSF and B7-1 gene-modified myeloma cell vaccine produces an in vivo antitumor immune response.Materia

14、ls and Methods Animals，myeloma cells and peripheral blood lymphocytes (PBL)In vivo experiments were performed in female NOD/SCID mice (from the Experimental Animal Center，Chinese Academy of Medical Sciences，Beijing)，aged 7 to 9 weeks，which were bred and maintained in specific pathogen-free condition

15、s.    In this study，a human myeloma cell line sko-007，with human leukocyte antigen (HLA) A2 positive，was kindly provided by Professor Bei-Fen SHEN from Beijing Institute of Basic Medical Sciences and was cultured in RPMI 1640 (Sigma) supplemented with 10% FBS，100 U/ml penicillin，

16、and 100 g/ml streptomycin.    PBLs of normal donors in the Beitaiping Road 2# Hospital of Beijing，China，applied for establishment of human immune system in NOD/SCID mice，were isolated by Ficoll-Paque separation as described previously. Since sko-007 cells are HLA-A2 positive，PBLs

17、 with the same HLA antigen were selected and cultured in RPMI 1640 containing 15% FBS，5% human AB sera，50 U/ml IL-2，100 U/ml penicillin，and 100 g /ml streptomycin. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37. HLA-A2 gene of PBLs was amplified by polymerase chain reaction

18、 (PCR) as described previously19. Briefly，genomic DNA was extracted from PBLs of normal donors according to Wizard? genomic DNA purification system (Promega，Madison，WI)，and HLA-A2 PCR was performed in a final volume of 50 l with the use of 500 ng DNA，0.1 nmol/L MgCl2，0.01 nmol/L dNTP，0.01 nmol/L eac

19、h primer，and 1 U Taq Polymerase (Promega) in PCR buffer. Five cycles，each consisting of 60 seconds at 96，60 seconds at 66，and 120 seconds at 72，followed by 25 cycles，each consisting of 60 seconds at 96，60 seconds at 56，and 120 seconds at 72，were performed in a Marstercycler Personal DNA Thermocycler

20、 (Eppendorf，Hamburg，Germany). The primers for HLA-A2 amplification were 5-CCTCGTCCCCAGGCTCT-3 (sense) and 5-TGGCCCCTGGTACCCGT-3 (antisense). -actin was used as internal standard. The reaction products were electrophoretically separated through a 1.5% agarose gel and stained with ethidium bromide. Th

21、e expected products generated by PCR were 813 base pairs (bp) and 396 bp for HLA-A2 and -actin，respecti-vely.    Recombinant adenovirus and gene transfer Ad-GFP (recombinant adenovirus expressing green fluorescence protein) was kindly provided by the Gene Therapy Unit，Baxter Heal

22、thcare Corp.，USA. Ad-p53/GM-CSF/B7-1 (recombinant adenovirus coexpressing human wild-type p53，GM-CSF and B7-1 proteins) was constructed by our department via homologous recombination in HEK293 cells (Ads E1-transformed human embryonal kidney cells). The inserted human wild-type B7-1 gene was driven

23、by a Rous sarcoma virus (RSV) promoter，and p53 and GM-CSF genes，linked by internal ribosome entry site (IRES)，were driven by a cytomegalovirus (CMV) promo-ter20. These two kinds of recombinant adenovirus with high titer and purity were obtained by large-scale amplification in HEK293 cells and ultra-

24、centrifugation in CsCl density gradient solution. The infection titers of Ad-GFP and Ad-p53/GM-CSF/B7-1 used in this study were 1×1011 pfu/ml and 5×1010 pfu/ml respectively.    To produce the myeloma cell vaccine，sko-007 cells were infected with Ad-GFP or Ad-p53/GM-CSF/

25、B7-1 at a multiplicity of infection (MOI) of 200 for 2 hours. Culture medium was used for mock infection. After an additional 48 hours incubation at 37，5% CO2，transgenic expression of GFP，as well as B7-1，GM-CSF and p53 mediated by adenovirus were determined by flow cytometry，ELISA and Western blot，r

26、espectively，as previous described12. Establishment of human immune system and vaccine administration Eighteen NOD/SCID mice were injected intraperitoneally with (3-4)×107 HLA-A2 PBLs in 0.5 ml PBS，pH 7.4 on day 0 and then were randomly divided into 3 groups: control，Ad-GFP and Ad-p53/GM-CSF/B7-

27、1 group. Each group of 6 huPBL-NOD/SCID mice was immunized twice subcutaneously on the abdomen with 1 ×106 irradiated Ad-p53/GM-CSF/B7-1- or Ad-GFP-infected sko-007 cells in 0.2 ml PBS or 0.2 ml PBS on days 7 and 14. Following vaccination，all animals received subcutaneous injection of 500 U rec

28、ombinant human IL-2 per mouse for 3 times a week until sacrificed. Tumor-challenge studies On day 7 after the second injection，various treatment groups of mice were challenged subcutaneously on their backs with 5 × 106 live sko-007 cells. Tumor growth was monitored 2 to 3 times a week by measur

29、ing 2 maximum diameters of the tumor at the site of challenge with a vernier caliper and was reported as a mean of the 2 diameters. Mice were weighed using an electric scale and sacrificed by eyeball extirpation on day 66. The tumors were excised and weighed. The tumor volume was determined by measu

30、ring the length (a)，width (b) and thickness (c) using a vernier caliper and calculated by the formula: abc/6(mm2). The weight index was calculated as the weight ratio of tumor / mouse. Processing of specimens for histopathology When mice were killed，tumor tissues were removed from various treatment

31、groups and fixed in 10% phosphate-buffered formalin for 24 to 48 hours，processed through graded alcohols，and embedded in paraffin. Serial sections of tumors were cut at various levels and stained with hematoxylin and eosin for histopathologic analysis. Additional sections were used for immunohistoch

32、emical staining for human T lymphocytes using monoclonal rabbit anti-human CD3 antibody (Zhongshan，Beijing). Subsequently，tissues were incubated with polyclonal biotin-conjugated goat anti-rabbit antibody (Zhongshan) followed by streptavidin- horseradish peroxidase (Zhongshan). The staining reaction

33、 was performed for 10 min with 3，3-diamino-benzidine-tetrahydrochloride (Zhongshan) in PBS (60 mg/100 ml). Finally，the tumor tissues were stained with hematoxylin to display the caryons. Determination of human Ig levels Levels of human IgG and IgA in the sera of huPBL-NOD/SCID mice were determined b

34、y agar diffusion assay. When mice were sacrificed，peripheral blood was harvested by eyeball extirpation. 100 l sera were aspirated following centrifugation and mixed with 300 l distilled water. 10 l mixture of ea     Results Discrimination of HLA-A2 PBLs and reconstruction of hum

35、an immune system in NOD/SCID mice To efficiently present tumor antigens of HLA-A2 positive sko-007 cells，PBLs from HLA-A2 normal donors were discriminated. As shown in Figure 1，3 of 6 DNA samples of PBLs were HLA-A2 positive，confirmed by PCR amplification. These PBLs from 3 donors were then inoculat

36、ed to establish human immune system in 18 NOD/SCID mice by intraperitoneal injection.    To testify the availability of the huPBL-NOD/SCID mice model，human Igs in the sera and human leukocytes in the spleens were determined at the time when animals were sacrificed. Human IgG cont

37、ents in control，Ad-GFP or Ad-p53/GM-CSF/B7-1 group were 5.73±3.14 g/L，5.42±2.11 g/L or 8.41±5?21 g/L，respectively (P>0.05)，and IgA contents were 1.29±0.30 g/L，1.39±0.46 g/L or 1.61±0.70 g/L，respectively (P>0.05). Human leukocytes were found in the spleens of mice

38、(Figure 2). Our data indicated that human immune system was successfully established in NOD/SCID mice. High expression of transgenes in sko-007 cells Applying GFP as the reporter gene，several research groups，including us，have demonstrated efficient adenovirus-mediated gene transfer into myeloma cell

39、s. At a MOI of 200 pfu/cell，nearly all sko-007 cells were GFP-positive without obvious adenoviral toxicity (Figure 3A). Our previous study further indicated that human wild-type p53，GM-CSF，and B7-1 genes media- ted by the recombinant adenovirus Ad-p53/GM-CSF/B7-1 were highly expressed in three MM ce

40、ll lines (sko-007，U266 and RPMI8226) and primary myeloma cells，and that Ad-p53/GM-CSF/B7-1 had growth-    inhibiting and apoptosis-inducing as well as immunogenicity-enhancing effect on myeloma cells12. In this study，we chose sko-007 to produce gene-modified MM vaccine and transp

41、lanted tumor in huPBL-NOD/SCID mice model. As shown in Figure 3B-D，the expressions of B7-1，GM-CSF and p53 on Ad-p53/GM-CSF/B7-1-infected sko-007 cells were determined by flow cytometry，ELISA and Western blot. Generation of Ad-p53/GM-CSF/B7-1-mediated immunity is protective against subsequent tumor c

42、hallengeTo examine whether the MM vaccine might affect its antitumor immunity in vivo，huPBL-NOD/SCID mice receiving 2 consecutive injections of irradiated (30 Gy) Ad-GFP- or Ad-p53/GM-CSF/B7-1-infected sko-007 cells or PBS were challenged with 5 ×106 sko-007 live tumor cells at day 7 after the

43、second injection. Tumor growth was followed at the injection site. There were no differences between 3 groups within 40 days after tumor cell injection. At the time of sacrifice，most animals in control or Ad-GFP group (4 of 6 mice with PBS and 5 of 6 mice with Ad-GFP-infected sko-007，respectively) d

44、eveloped massive local tumors. In contrast，only 3 of 6 animals injected with Ad-p53/GM-CSF/B7-1-transferred sko-007 cells had measura-ble tumors (Figure 4). SAS analysis of the tumor weight index showed that Ad-p53/GM-CSF/B7-1-infected sko-007 vaccination significantly reduced tumor growth，compared

45、with controls receiving Ad-GFP modified tumor cells or PBS (P<0.05 and P<0?001，respectively) (Figure 4).    The in vivo antitumor activity of Ad-p53/GM-CSF/B7-1- transferred sko-007 cells was further stu-died by histopathologic analysis of implanted tumors of 3 groups. In P

46、BS group，tumor cells were eugonic and showed typical morphologic features of neoplastic plasma cells，ie，irregular nuclear profiles with priminent nucleoli and abundant cytoplasma. Sheet necrotic areas were also identified (Figure 5A). Otherwise，in Ad-p53/GM-CSF/B7-1 group，tumor tissues displayed dif

47、fuse necrosis，mainly caused by apoptosis，accompanied with significant fibroplasias and blood vessel hyperplasia (Figure 5C). Tumor tissues of Ad-GFP group were similar to those of PBS group，accompanied with various degree of fibroplasias (Figure 5B). The results indicated that vaccination of mice wi

48、th Ad-p53/GM-CSF/B7-1-transferred sko-007 cells displayed enhanc    In this report，we have shown that vaccination of MM cells modified by human wild-type p53，GM-CSF and B7-1 genes resulted in protection against challenge by additional local injection of nontransfected parental tu

49、mor cells in NOD/SCID mouse model. Tumor tissues increasingly displayed diffuse necrosis，accompanied with significant fibroplasias and blood vessel hyperplasia. Human T cells inhabited in the spleens and infiltrated the tumor tissues. This local effect is systemically immune mediated，because vaccina

50、tion inhibited tumor growth from a subsequent tumor challenge at a distal site，which may particularly favor the antitumor responses required to treat the metastasis and spreading of human tumors. As showed in our previous study，this effect is probably mediated by the cytotoxicity of human lymphocyte

51、s inoculated intraperitoneally to NOD/SCID mice beforehand，which were activated by Ad-p53/GM-CSF/B7-1-transferred sko-007 cell vaccines.    In conclusion，we have shown that transgenic p53，GM-CSF and B7-1 expression induces a protective response against myeloma. The myeloma cell v

52、accine modified by these 3 genes may be of therapeutic value and can be considered for a phase I clinical trial of MM patients.    【參考文獻(xiàn)】 1Kyle RA. The role of high-dose chemotherapy in the treatment of multiple myeloma: a controversy. Ann Oncol，2000；11(Suppl 1): 55-582Chiusolo P

53、，Sica S，Piccirillo N，et al. Molecular and clinical following-up after stem cell transplantation for multiple myeloma. Ann Hematol，2001；80: 90-953Gahrton G，Bjorkstrand B. Progress in haematopoietic stem cell transplantation for multiple myeloma. J Intern Med，2000；248: 185-2014Liu Q，Gazitt Y. Adenovir

54、us-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34 hematopoetic progenitor cells and normal lymphocytes. Exp Hematol，2000；28: 1354-13625Trudel S，Li Z，Dodgson C，et al. Adenovector engineered interleukin-2 expressing autologous plasma cell vaccination after high-dose chemotherapy for multiple myeloma ? a phase I study. Leukemia，2001；15: 846-8546Tarte K，Zhang XG，Legouffe E，et al. Induced expression of B7-1 on myeloma cells following retroviral gene transfer results in tumor-specific recognition by