| Approximately 40,000 new cases of kidney cancer will be diagnosed and about 13,000 people, including adults and children, will die from this disease in 2006 in the United States. Renal cell carcinoma (RCC), the most common type of kidney cancer, accounts for more than 90% of malignant kidney tumors. Metastatic RCC (mRCC) has historically been refractory to conventional chemotherapy and chemotherapy. A minority of patients has shown some response to cytokine therapy with interleukin-2 (IL2) or interferon (IFN)-alpha, but associated toxicities can be severe. The studies demonstrated the occure and development of the cancer was related with the functional defection of T lymphocyte, and the further studies found the reason of which was the down-regulation of the mature dendritic cells (DC) by the body. In vitro, many experiments verified that the lymphocyte isolated from the advance stage cancer patient's peripheral blood still maintained very good reaction to the outside stimulation. So through culturing the DC in vitro and inducing the specific and high biology activity cytotoxic T lymphocyte (CTL), the injected back into the patient, which maybe becoming excellent therapy for the cancer. Therefore, if we know the target antigen for the cancer, how can we get the good biology activity and large amount specific CTL?DC is the most effective Antigen Presenting Cell at activating naive T cells, which can activate antigen specific CTL with the antigen successful loading. At present, granulocyte-macrophaen colony stimulating factor (GM-CSF) and Interleukin-4 (IL-4) can promote the generation of large numbers of Dendritic Cells from peripheral blood monocytes. However, the focus point of the protocal in pulsing DC and inducing CTL in vitro is how to loading the antigen effectively?The study showed: with different antigen loading technic, the DC activating efficiency is different. The CTL activity is also different. There are several ways to load the antigen, the popular strategy are: the antigen peptide transducing DC and virus vector carrying the antigen gene transfecting the DC.Tuansducing DC with antigen peptide has advantages such as antigen peptide is specific and which can immunize times, but which also has one main problem is the half-life of this kind of antigen peptide usually us very short, so only after several times of immunization can we get CTLwith good activity. Moreover, the transduction efficiency is low.Transfection DC with virus vector carrying antigen gene can make the DC produce a constant and large amount of antigen peptide, which can resolve the problem of antigen peptide transducing DC directly. At present, the common virus vectors for the DC transfection are: retrovirus (Retrov), adenovirus(Ad), et al. Ad vectors are the most currently used in clinical trails. Ad vectors are the Ad type 5 recombinant vectors which E1 region are removed. As we know, genes in the E1 region are necessary for activation of viral promoters and expression of both early and late genes. Thus, removal of the E1 coding sequence results in viruses that are severely impaired in their ability to replicate. Many features of adenovirus make it well suited for gene delivery, including the ability to grow recombinant viruses to high titers, a relatively high capacity for transgene insertion, and efficient transduction of both quiescent and actively dividing cells, usually without incorporation of viral DNA into the host cell genome. These characteristics, as well as the development of many methods for manipulating the viral genome, have made adenovirus a popular choice as a gene delivery vehicle. This is evidenced by the fact that adenovirus is currently being used in roughly one-quarter of all gene therapy clinical trials, making it second only to the use of retroviralvectors.Three parts of gene therapy study using Ad as vector were designed on the base of pre-studies. The first part is: An in vitro study of specific antitumor immunity induced by dendritic cells pulsed with kidney tumor association antigen G250 protein. The second part is: Generating the CTL by G250 gene transfecting DC to treat kidney tumor. The third part is: Immue response induded by DC transfected by G250 gene in vitro inhibit growth of implanted tumor and reject challenge of tumor cells in nudes. Through them to study the possibility of kidney TAA G250 protein pulsing DC and G250 antigen gene transducing DC generating the CTL in kidney tumor treatment. Furthermore, we will optimize the protocol and make it as the experiment base for the future clinical trial in some kinds of cancer with clear antigen.The first part: An in vitro study of specific anti-kidney cancer immunity induced by dendritic cells pulsed with G250 protein.1. Methods1.1 Generation of monocyte-derived DCs from peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Paque density gradient centrifugation of heparinized blood obtained from healthy donors and washed twice in PBS. The PBMCs were inoculated into six well culture plates at 37℃under a 5% CO2 atmosphere for 4～6 hours, the adherent cells were incubated in AIM-V Medium containing rhIL-4 (final concentration of 1000IU/ml)and rhGM-CSF (final concentration of 800IU/ml), and the nonadherent cells was frozen as effective cell for use at a later date. On day 5, when they were defined as iDC, the adherent cells were divided into two groups: one was protein pulsed group, the other was control group. The protein pulsed group was pulsed with G250 protein (750 mg/ml). The control group was pulsed with PBS solution. At the same time, cytokines TNF-αat a final concentration of 200IU/ml was include in the medium in both groups throughout the culture. Both groups were cultured for a further 3 days. Half of the medium was replaced with fresh medium every other day. DCs were harvested on day 8, and they were defined as mDC.1.2 Phenotypic analysis of DCs by flow cytometry.On day 8, the phenotypic analysis of DCs was carried out by immunofluorescence staining of dendritic-monocyte differentiation and activation markers. Cells were labelled with human monoclonal antibodies (mabs) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The following FITC-conjugated mabs were used: anti-HLA-DR, anti-CD83, anti-DC40. The following mabs were used as PE-conjugated reagents: anti-CD86, anti-CD80. anti-CD1a. PE/FITC isotype-matched mouse immunoglobulins were used as negative controls. Cells were washed once with PBS and incubated with the appropriate mab for 30 min on ice. Then cells were washed once again and fixed in PBS/1% paraformaldehyde. Samples were acquired and analyzed by flow cytometry.1.3 Isogenic mixed lymphocyte reactionAfter the two different group DCs were incubated with mitomycin C (30ug/ml) for 30 minutes, and then the DCs were washed with PBS solution. Lymphocytes were added into a 96-well culture plate. as responder cells at a concentration of 1x105/ml. DCs were collected in each group as stimulator cells at various ratios of stimulator cells to responder cells( 1: 5,1: 10,1: 20). The total terminal volume was 200μl per well. Each group was set to triple wells. After co-culture for 5 days, Add 20μl MTT (5g·L-1) to each well of multiwell culture plate, incubate for 4 hours, then add 100μL DMSO, mixed about 10 min until the crystal completely dissolved. The absorption value (OD value) of each well was immediately read by automatic enzyme linked detector at 570nm wavelength.1.4 Analysis of CTL for cytotoxicity essaysOn day 8, nonadherent PBMCs from the same healthy donors were washed and added to protein-pulsed or mock-treated DCs (ratio of 20:1, responders: dendritics) in six-well culture plates. The RPMI-1640 cultures were supplemented with recombinant human GM-CSF(800U/ml), recombinant human IL-2(10U/ml), and IL-7(20ng/ml). After 5 days of coculture (day 13), the cells were collected for the CTL activity assay.Then, we made three group effective CTLs: In group 1, CTLs were T+Ad/G250-DC; In group 2, CTLs were T+Protein/G250-DC CTLs; In groups 3, CTLs were T+N-DC. Part of the three group CTLs were carried out for Phenotypic analysis by flow cytometry. The Cells were labelled with human monoclonal antibodies (mabs) FITC or PE. Each group of CTLs as stimulator cells and 786-0 or A549 as responder cells were coculmred in a 96-well culture plate for 48 hours at various ratios of effector cells to target cells( 5: 1,10: 1,20: 1,40: 1); The total terminal volume was 200μl per well. Each group was set to triple wells. Add 20μl MTT (5g·L-1) to each well of multiwell culture plate, incubate for 4 hours, then add 100μL DMSO, mixed about 10 min until the crystal completely dissolved. The absorption value (OD value) of each well was immediately read by automatic enzyme linked detector at 570nm wavelength.The killing activity was measured by MTT and the killing rate was calculated by the follow formula:Killing rate=[1-ODexperiment group/ODresponder cells group+ODtarget cells group]x100%.1.5 Statistical analysisThe data were analyzed by the SPSS13.0 statistical software. Statistical method used analysis of variance of factorial design date, and the interblock difference compare applied LSD. P≤0.05 was considered to be statistically significant.2. Results(1). There were high expression of CD40, CD80, CD83, CD86 and HLA-DR, and low expression of CD1a in both the groups. (2). The ability to elicit lymphocyte proliferation in the DC pulsed with G250 protein(DC-G250)group was strong (F=83.545, P=0.000), whereas in the DC pulsed with PBS(DC-N) as control group it was low. At different ratio of effector cells to target cells, a significant difference was observed between the two groups (F=23.273, P=0.000). (3). The percentage of CD3+CD56+ and CD8+ cells in total CTL cells significantly increased in DC-G250 group. Phenotype of CTLs induced by DC-G250 higher than DC-N group (t=9.589 P=0.01; t=11.806 P=0.000). (4). In vitro the cytotoxicity to 786-0 of T lymphocytes activated by G250 protein pulsed DCs were greater than those of T lymphocytes in the control group (F=29.483, P=0.000). The cytotoxicity to A549 of T lymphocytes activated by G250 protein pulsed DCs were no significant difference than those of T lymphocytes in the control group.3 ConclusionsThe kidney tumor association antigen G250 pulsed DC vaccines can induce an effective and specific anti-renal carcinoma effect. The TAA G250 antigen may have a major impact on the adoptive immunotherapeutic protocols for patients with kidney cancer. The second part: Study on Generating the CTL by G250 gene transfecting DC using Ad as vector to treat kidney cancer.1. Methods1.1 Ad/G250 transfecting the DC and generating the CTLPeripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Paque density gradient centrifugation of heparinized blood obtained from healthy donors and washed twice in PBS. The PBMCs were inoculated into six well culture plates at 37℃under a 5% CO2 atmosphere for 4～6 hours. After the removal of the nonadherent cells, the adherent cells were divided into three groups; one was gene transfer group; one was protein pulsed group; another one was control group. The gene transfer group was infected with Ad/G250 virus. After 5 hours of incubation, the medium/virus solution was removed, and the fresh AIM-V medium was added. GM-CSF at a final concentration of 800IU/ml was included in the medium in three groups throughout the culture. Human IL-4 at 1000IU/ml was added at day 3. On day 5, the protein pulsed group was pulsed with G250 protein (750mg/ml). At the same time, the control group was pulsed with PBS solution., cytokines TNF-αat a final concentration of 200IU/ml was include in the medium in the three groups throughout the culture. The three groups were cultured for a further 3 days. On day 8, they were defined as mDC. On day 8, nonadherent PBMCs from the same healthy donors were washed and added to Ad/G250-loaded, protein-pulsed or mock-treated DCs (ratio of 20:1, responders: dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF(800U/ml), recombinant human IL-2(10U/ml), and IL-7(20ng/ml). After 5 days of co-culture (day 13), the cells were collected for the CTL activity assay.1.2 The G250 mRNA test, the DC surface marker analysis.On day 8, collected the suspend cells(Mature DC), observed the morphologic features of DC with light microscope. G250 mRNA expression was detected in transduced DCs using RT-PCR amplification, and the unpulsed-DC served as control. The resulting G250 RT-PCR products were observed. Cell surface marker analysis of DCs was by flow cytometry. For the characterization of DCs, a panel of surface marker was used: CD83,CD80,CD86,HLA-DR,CD1a.1.3 The immunity activity testing of G250-specific CTLsCell surface marker analysis of stimulated T cells: The primed T-cell populations were analyzed for surface markers on day 13. A panel of mAbs recognizing the following antigens was used: PE-anti-CD4, FITC-anti-CD8, and PE-anti-CD56. Thus, the ratio of CD8/CD4 and CD8/CD56 was analysed with the flow cytometry.Analysis of CTL for cytotoxicity assays: The primary kidney cancer cell line 786-0(HLA-A2 haplotype) was used as one G250-positive target. The HLA haplotypes of all donors were compatible with this cell line. Lung cancer cell line A549 was used as negative target. The cells were used for cytotoxicity assays by MTT method.2. ResultAd/G250 transfected DCs successful. Ad/G250-loading of DCs resulted in: (1)G250 experssion in DCs. (2). RT-PCR amplificated G250 mRNA in Ad/G250 transfecting DCs. (3) high CD80,CD83,HLA-DR and CD86 experssion in DCs. (4) high CD8: CD4 and CD8: CD56 T cell ratios. (5) strong, rapid G250-specific CTLs. In vitro the cytotoxicity to 786-0 of T lymphocytes activated by Ad-G250 transfecting DCs were greater than those of T lymphocytes in the protein G250 group and control group. The CTL stimulated by Ad-G250 group had much higher cytotoxicity to renal carcinoma cell line 786-0, as compared with A549.3. ConclusionThese date suggest that Ad-loading of DCs may be useful for immunotherapeutic protocols against self-antigens and that the G250 antigen is a potentially appropriate target for cell-mediated immunotherapeutic treatment for the kidney cancer.The third part: Immune response induced by DC transfected by G250 gene in vitro inhibit growth of implanted tumor and reject challenge of tumor cells in nudes.1. Methods1.1 Generation of monocyte-derived DCs from peripheral blood mononuclear cells Peripheral blood was derived from healthy donors. Ficoll gradient-purified PBMCsWere inoculated into six well culture plates for 4～6 hours, and the adherent cells were selected following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent cells were divided into two groups: one was gene transfer group; one was protein pulsed gruoup. The gene transfer group was infected with Ad/G250 virus. After 5 hours of incubation, the medium/virus solution was removed, and the fresh AIM-V medium was added. GM-CSF at a final concentration of 800IU/ml was included in the medium in two groups throughout the culture. Human IL-4 at 1000IU/ml was added at day 3. On day 5, the protein pulsed group was pulsed with G250 protein (750 mg/ml). At the same time, cytokines TNF-αat a final concentration of 200IU/ml was included in the medium in the three groups throughout the culture. The two groups were cultured for a further 3 days. On day 8, they were defined as mDC.1.2 The two groups CTLs generated by the two groups DCs.On day 8, the T lymphocytes from the same healthy donors were added to Ad/G250-loaded DCs and protein-pulsed DCs. (ratio of 20:1, responders: dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF(800U/ml), recombinant human IL-2(10U/ml), and IL-7(20ng/ml). After 5 days of coculture, the cells were collected for inhibiting growth of implanted tumor in nudes mice.1.3 Animals and establishment of the xenograft RCC model in nude miceFemale BALB/c-nu/nu mice were purchased and were age-matched (4 weeks of age) at the beginning of each experiment. They were maintained in specific pathogen-free (SPF) facilities at our institute. For tumor implants, 786-0 cells (2 x 106) in 200μl of PBS were injected s. c. into the right side of back of female nude mice 4 weeks of age. Tumor dimensions were measured with digital calipers to obtain two diameters of the tumor formed. The tumor incidence and tumor size were calculated with the equations shown below. Tumor size (16 days after inoculation) was calculated using the formula as follows: Tumor size =a x b, where a is the longest diameter and b is the shortest.1.4 Immune response induced by DC transfected by G250 gene and pulsed with G250 protein in vivo prevent growth of implanted tumor in nude miceThe experimental mice were assigned randomly to three groups (n=5): control group, CTL-DC-Ad/G250 group and CTL-DC-Pro/G250 group. Mice in the control were inoculated subcutaneously with normal saline (200ul/each) into the right side of back of athymic nude mice simultaneously. Nude mice in the CTL-DC-Ad/G250 groups were treated with CTL (2x106 cells/each)induced by DC-Ad/G250 subcutaneously. Nude mice in the CTL-DC-Pro/G250 group were treated with CTL (2x106 cells/each) induced by DC-Pro/G250 subcutaneously. 72 hours after inoculation, 786-0(2x106 cells/each) were inoculated subcutaneously into the other side of back of athymic nude mice in the three groups simultaneously. From then on, the growth of implant tumor was observed every three day for 30 days.1.5 Immune response of CTL induced by DC transfected by G250 gene in vivo inhabit growth of implanted tumor nude mice On 16 days after inoculation in 1.3, there were 12 nude mice growing the xenograft. On 21 days after inoculation, 10 nude mice, that xenograft average size had reached 8.6±1.2mm2, were selected for next experiments, another 2 nude mice, that xenograft size growed largerly than others, and 3 not growing xenograft were rejected from experiments. The 10 growing xenograft mice were assigned randomly to two groups (n=5): Control group and CTL-DC-Ad/G250 group. Mice in the control were inoculated s. c. with normal saline (200ul/each) into the other side of back of nude mice. Mice in the CTL-DC-Ad/G250 groups were treated with CTL (2x106 cells/each) induced by DC-Ad/G250 subcutaneously into the other side of back of nude mice. From then on, the growth of implant tumor was observed every three day until that day when the mice have been inoculated 45 days.2. Results2.1 Happenning rate of xenograft tumorOn the 30th day, there were no xenograft tumor growth in CTL-DC-Ad/G250 group and CTL-DC-Pro/G250 group; but the control group had 4 mice growing the xenograft tumor. The tumor formation incidences of 786-0 cells in the control group (n=5) were 80%.2.2 Inhibitory growth of implanted tumor nude miceOn the 45th day of inoculation, we finished the experiments about the xenograft in nude mice. The mean volume of tumor of control group was 143.37±11.6 mm2, whereas that of CTL-DC-Ad/G250 was 42.55±5.62mm2 (t=2.355, P=0.047＜0.05)Evidently, the CTL induced by DC transfected by Ad/G250 could prevent the growth of xenograft tumor, compared with the control group.3. ConclusionsThe CTL induced by DCs transfected by Ad/G250 could not only preclude the growth of xenograft tumor in nude mice and prevent the mice against the further challenge of 786-0 tumor cells, but also inhibit the growth of implanted tumor. The result suggest, as a new concept anti-tumor vaccine, DC transfected by Ad/G250 or pulsed by G250 protein may play an important role in therapy and prevent against kidney cancer.
|
PDF Full Text Request