In vivo antitumor effect of CD40L-transduced tumor cells as a vaccine for B-cell lymphoma

CD40-CD40 ligand (CD40L) interactions play a critical role in the activationof cellular immunity. CD40L enhances the antigen presentation function of CD40-expressing B cells. We have used a murine B-cell lymphoma model (A20) to study the in vivo antitumor effect of the administration of tumor cells transduced with a recombinant adenovirus encoding CD40L (AdvCD40L). After infection with AdvCD40L, A20 tumor cells up-regulate several T-cell costimulatory molecules (CD80, CD86, ICAM-1, and LFA-3) and Fas expression. Animals vaccinated with irradiated tumor cells transduced with AdvCD40L are protected against a lethal dose of parental A20 tumor cells. Animals with pre-existing tumors treated with AdvCD40L-transduced tumor cells display inhibition of the tumor growth, and this treatment confers a survival advantage. In vivo depletion studies demonstrate that both CD4(+) and CD8(+) T cells mediate the antitumor immunity provided by AdvCD40L-transduced tumor cells. These results show that genetic modification of tumor B cells with CD40L can be a useful strategy to promote systemic immunity against B-cell malignancies and provide an in vivo system to allow for additional evaluation and refinement of this approach.     

Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects

Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conventional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule transgenes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowlpox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccinations with rF-CEA or two vaccinations with rF-CEA/B7-1. Moreover, up to four vaccinations with rF-CEA/TRICOM induced greater CEA-specific T-cell responses with each vaccination. (b) A diversified prime and boost strategy using a prime with a recombinant vaccinia vector expressing CEA and the triad of costimulatory molecules (designated rV-CEA/TRICOM) and a boost with rF-CEA/TRICOM was more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA/TRICOM alone. (c) The addition of granulocyte macrophage colony-stimulating factor (GM-CSF) to the rF-CEA or rF-CEA/TRICOM vaccinations via the simultaneous administration of a rF-GM-CSF vector enhanced CEA-specific T-cell responses. These strategies (TRICOM/diversified prime and boost/GM-CSF) were combined to treat CEA-expressing carcinoma liver metastases in CEA-transgenic mice; vaccination was initiated 14 days posttumor transplant. Antitumor effects in terms of survival and CD8(+) and CD4(+) responses specific for CEA were also observed in this CEA-transgenic mouse model. These studies demonstrate that the use of cytokines and diversified prime and boost regimens can be combined with the use of recombinant vectors expressing signal 1 and multiple costimulatory molecules to further amplify T-cell responses toward more effective vaccine strategies.     

重组鼠CD40配体的Ankara痘病毒疫苗对鼠淋巴瘤的免疫治疗作用的研究

目的研究Ankara痘病毒和mCD40L的重组体TBC-MVA-mCD40L对小鼠淋巴瘤的免疫治疗作用。方法 应用流式细胞术检测经体外或者体内转染mCD40L的A20瘤细胞的表面共刺激分子CD80和CD86的表达。构建了BALB/c小鼠的B细胞淋巴瘤模型并分组接受瘤内注射TBC-MVA-mCD40L联合系统化疗的治疗模式。另外, 应用与A20细胞孵育后的TBC-MVA-mCD40L对鼠进行免疫, 观察其对鼠淋巴瘤的预防作用。通过观察小鼠接种的肿瘤的大小和生长情况来判断治疗效果。结果 转染CD40L后流式检测A20细胞表面共刺激分子CD80和CD86的表达增强。TBC-MVA-mCD40L免疫治疗联合化疗能成功地使肿瘤消退并使荷瘤鼠获得长期生存。TBC-MVA-mCD40L免疫预防接种能使小鼠获得长期的抗肿瘤免疫。结论 对B淋巴瘤细胞的CD40L基因修饰策略能有效增强机体的对抗B细胞淋巴瘤的系统免疫, 为将来B细胞淋巴瘤患者肿瘤内注射TBC-MVA-mCD40的免疫治疗和预防方法提供依据, 为该免疫治疗方法的临床应用奠定基础。     

A triad of costimulatory molecules synergize to amplify T-cell activation

The activation of a T cell has been shown to require two signals via molecules present on professional antigen-presenting cells: signal 1, via a peptide/MHC complex; and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were used because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results using the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell:T-cell ratios. Experiments using a four-gene construct also showed that TRICOM recombinants can enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate for the first time the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development.     

Induction of an antigen cascade by diversified subcutaneous/intratumoral vaccination is associated with antitumor responses.

PURPOSE: Cancer vaccines targeting tumor-associated antigens are being investigated for the therapy of tumors. Numerous strategies, including the direct intratumoral (i.t.) vaccination route, have been examined. For tumors expressing carcinoembryonic antigen (CEA) as a model tumor-associated antigen, we previously designed poxviral vectors that contain the transgenes for CEA and a triad of T-cell costimulatory molecules, B7-1, intercellular adhesion molecule-1, (ICAM-1), and leukocyte function associated antigen-3 (LFA-3) (CEA/TRICOM). Two types of poxvirus vectors were developed: replication-competent recombinant vaccinia and replication-defective recombinant fowlpox. We have shown previously that a vaccine regimen composed of priming mice s.c. with recombinant vaccinia-CEA/TRICOM and boosting i.t. with recombinant fowlpox-CEA/TRICOM was superior to priming and boosting vaccinations using the conventional s.c. route in inducing T-cell responses specific for CEA. These studies also showed that CEA was needed to be present both in the vaccine and in the tumor for therapeutic effects. EXPERIMENTAL DESIGN: To determine specific immune responses associated with vaccination-mediated tumor regression, CEA-transgenic mice bearing CEA(+) tumors were vaccinated with the CEA/TRICOM s.c./i.t. regimen, and T-cell immune responses were assessed. RESULTS: In CEA(+) tumor-bearing mice vaccinated with the CEA/TRICOM s.c./i.t. regimen, T-cell responses could be detected not only to CEA encoded in vaccine vectors but also to other antigens expressed on the tumor itself: wild-type p53 and an endogenous retroviral epitope of gp70. Moreover, the magnitude of CD8(+) T-cell immune responses to gp70 was far greater than that induced to CEA or p53. Finally, the predominant T-cell population infiltrating the regressing CEA(+) tumor after therapy was specific for gp70. CONCLUSION: These studies show that the breadth and magnitude of antitumor immune cascades to multiple antigens could be critical in the therapy of established tumors.     

Use of adenoviruses encoding CD40L or IL-2 against B cell lymphoma

Some B cell lymphomas lack important costimulatory properties that could prevent them from being used as cell based vaccines. Infection of A20 B lymphoma cells with a replication-defective adenovirus encoding murine (m) CD40L, but not mIL-2, produces an antigen presentation phenotype with upregulation of MHC Class I/II, induction of B7-1/2 molecules and production of MIL-12 and MIP-1alpha. Subcutaneous vaccination with irradiated Ad-mCD40L-infected- or Ad-mIL-2-infected-A20 cells generated A20-specific CD8+ T cell responses and cross reactive A20 Ig antibodies. Only vaccination with Ad-mCD40L-infected A20 cells produced a significant delay in tumor growth and long-term survival (p = 0.0039). Stronger protective immunity to A20 challenge was generated by intravenous priming with A20 cells infected with Ad-mCD40L, Ad-mIL-2 or their combination followed by a boost immunization with A20 cells activated with syngeneic fibroblasts expressing CD40L. Compared to Ad-LacZ-infected A20 priming, the combination priming was most effective followed by Ad-mCD40L and Ad-mIL-2 (p = 0.0027, p = 0.0027, p = 0.0163 respectively). Significant A20-specific CD8+ T cell-mediated cytotoxicity was only demonstrated in splenocytes from these groups of vaccinated animals. By contrast, ELISPOT assay of splenocytes from all A20 prime/boosted vaccinated groups demonstrated increases in gamma-interferon release by T cells elicited by in vitro stimulation either with A20 cells or another syngeneic 2PK-3 lymphoma, indicating the presence of cross reactive immunity. Similarly anti-A20 immunoglobulin antibodies generated after vaccination were not necessarily A20 idiotype-specific. Direct therapy of pre-established tumors was achieved with the combination of Ad-mCD40L and Ad-mIL-2 given at Days 4 and 8 at the tumor site with a significant long-term survival of 85% of tumor-bearing mice (p = 0.0001). Our study strongly supports the use of Ad-CD40L and Ad-IL-2 combination therapy for the treatment of patients with B cell lymphoma.     

Amplification of the lytic potential of effector/memory CD8+ cells by vector-based enhancement of ICAM-1 (CD54) in target cells: implications for intratumoral vaccine therapy

We demonstrated that enhanced expression of the costimulatory molecules CD80, CD54 and CD48 (designated rF-TRICOM) on target cells, as delivered via a recombinant fowlpox vector, results in an increased state of stimulation of CD8+ T cells, and consequent increased lysis of target cells. CTL studies in conjunction with antibody-blocking studies demonstrated that the enhanced effector activity of these CD8+ T cells is mediated mainly through CD54. Intracellular staining of CD8+ cells that interact with target cells infected with rF-TRICOM showed that they contain higher amounts of perforin and have a higher level of perforin message. Enhanced expression of costimulatory molecules (specifically CD54) on target cells using rF-TRICOM vectors also leads to the formation of stable conjugates/synapses between targets and T cells. The interaction of T cells with target cells that overexpress costimulatory molecules upon infection with rF-TRICOM leads to enhanced signaling through Lck, ZAP70, and STAT-1 in CD8+ T cells and heightened lytic activity of CD8+ cells through the formation of a greater number of immunological synapses. This, in turn, leads to enhanced signaling in T cells. Finally, studies were conducted in mice in which CEA is a self-antigen in an attempt to understand the potential clinical relevancy of intratumoral vaccine therapy. Mice were transplanted subcutaneously with CEA expressing tumors. Intratumoral (i.t.) vaccination was administered 8 days post tumor transplant. Mice vaccinated i.t. with rF-TRICOM demonstrated significantly reduced tumor growth and 40% of the mice had complete tumor regression. The antitumor effects were further improved by the addition of tumor antigen (CEA) in the vaccination by utilizing rF-CEA/TRICOM, with 80% of the mice experiencing complete tumor regression. These studies thus support the concept of intratumoral vaccination employing vectors expressing costimulatory molecules.     

Analyses of recombinant vaccinia and fowlpox vaccine vectors expressing transgenes for two human tumor antigens and three human costimulatory molecules.

PURPOSE: The poor immunogenicity of tumor antigens and the antigenic heterogeneity of tumors call for vaccine strategies to enhance T-cell responses to multiple antigens. Two antigens expressed noncoordinately on most human carcinomas are carcinoembryonic antigen (CEA) and MUC-1. We report here the construction and characterization of two viral vector vaccines to address these issues. EXPERIMENTAL DESIGN: The two viral vectors analyzed are the replication-competent recombinant vaccinia virus (rV-) and the avipox vector, fowlpox (rF-), which is replication incompetent in mammalian cells. Each vector encodes the transgenes for three human costimulatory molecules (B7-1, ICAM-1, and LFA-3, designated TRICOM) and the CEA and MUC-1 transgenes (which also contain agonist epitopes). The vectors are designated rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM. RESULTS: Each of the vectors is shown to be capable of faithfully expressing all five transgenes in human dendritic cells (DC). DCs infected with either vector are shown to activate both CEA- and MUC-1-specific T-cell lines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOM vectors. Thus, no evidence of antigenic competition between CEA and MUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM are also shown to be capable of generating both MUC-1- and CEA-specific T-cell lines; these T-cell lines are in turn shown to be capable of lysing targets pulsed with MUC-1 or CEA peptides as well as human tumor cells endogenously expressing MUC-1 and/or CEA. CONCLUSION: These studies provide the rationale for the clinical evaluation of these multigene vectors in patients with a range of carcinomas expressing MUC-1 and/or CEA.     

Rejection of murine melanoma-cells transfected with the intercellular-adhesion molecule-1 gene

The effects of intercellular adhesion molecule-1 (ICAM-1) expression on-tumor rejection and induction of antitumor immunity in a murine melanoma system was studied. K1735 melanoma cells genetically engineered to express the murine ICAM-1 gene were rejected in immunocompetent hosts, and that this rejection was mediated by CD4(+) as well as by CD8(+) T lymphocytes. We also found that ICAM-1 transfected tumor cells provided costimulatory signals to both CD4(+) and CD8(+) T cells in vitro. In addition, immunization of mice with K1735-ICAM-1 transfectants induced protective immunity against the parental ICAM-1-negative tumor. Our findings suggest that ICAM-1 expression on nonimmunogenic tumor cells causes tumor rejection and augments tumor-specific immunity.     

Vector-based vaccine/cytokine combination therapy to enhance induction of immune responses to a self-antigen and antitumor activity

Many antigens associated with human tumors are overexpressed in tumor cells as compared with normal tissues; these "self" tumor-associated antigens are also expressed during fetal development, and it is, thus, not surprising that they are either weakly immunogenic or functionally nonimmunogenic in the tumor-bearing host. In the studies reported here, we have used different vaccines and vaccine strategies in an attempt to develop antitumor immunity in a stringent animal model. The tumor antigen used was human carcinoembryonic antigen (CEA). The model used was CEA transgenic mice, in which the human CEA transgene is under the control of the endogenous CEA promoter; CEA is expressed in fetal tissues and normal gastrointestinal tissues, and CEA protein is found in sera. Previous studies have shown these CEA transgenic mice to be tolerant to the induction of CEA immunity using CEA protein in adjuvant as an immunogen. CEA-expressing tumor cells were implanted 14 days before vaccine therapy. The vaccines used were recombinant vaccinia virus containing the transgenes for CEA and three T-cell costimulatory molecules [B7-1, ICAM-1, and LFA-3, designated recombinant vaccinia (rV)-CEA/TRICOM], with each transgene under the control of individual poxvirus promoters, and a replication-defective avipox virus (fowlpox; rF) containing the same four transgenes (designated rF-CEA/TRICOM). The results demonstrate that (a) continued boosting with vaccine is required to maintain CEA-specific T-cell responses, and boosting with rF-CEA/TRICOM is superior to boosting with rF-CEA; (b) a diversified vaccination protocol consisting of primary vaccination with rV-CEA/TRICOM followed by boosting with rF-CEA/TRICOM is more efficacious than homogeneous vaccination with rF-CEA/TRICOM in the induction of both CEA-specific T-cell responses and antitumor activity; and (c) the use of cytokines, local granulocyte macrophage colony-stimulating factor (GM-CSF) and low-dose systemic interleukin 2, in combination with vaccine is essential in inducing antitumor activity, as compared with the use of cytokines alone, or the use of vaccines without cytokine. Both GM-CSF and interleukin 2 were shown to contribute to the induction of CEA-specific T-cell responses. These studies thus provide a "proof of concept" that potent vaccines and vaccine strategies, in combination with cytokines, may be essential to obtain the level of T-cell responses directed against a self-antigen that is necessary to achieve antitumor responses.     

B7-2 expression above a threshold elicits anti-tumor immunity as effective as interleukin-12 and prolongs survival in murine B-cell lymphoma

The costimulatory molecule, B7-2, is expressed by various lymphomas, but this level of expression is not sufficient to generate effective anti-tumor immunity in vivo. To determine whether up-regulated expression of the costimulatory molecule, B7-2, leads to more effective anti-tumor immunity in vivo, the A20 murine model of B-cell lymphoma was used. A20 tumor cells express major histocompatibility complex (MHC) I and II molecules and moderate constitutive levels of B7-2. While B7-1 and B7-2 have been introduced into tumor cells lacking these molecules, studies have not been conducted to determine whether tumors that constitutively express B7-1 or B7-2 can be made more immunogenic by increasing the expression of these molecules. In this report, A20/B7-2 transfectants expressing greater levels of B7-2 were rejected in syngeneic mice, and systemic immunity against the A20 parental cells was generated. Treatment with the A20/B7-2 variant cells significantly improved the survival of tumor-bearing mice. Coinjection with IL-12 secreting variants did not further augment the anti-tumor immunity observed for B7-2 therapy alone. Both CD8(+) T cells and natural killer (NK) cells mediated the anti-tumor immune response observed in A20/B7-2 immunized mice. In mice that developed tumors after immunization with the A20/B7-2 variant cells, resected tumor cells were shown to express lower levels of B7-2 than the transfected variants. These results suggest that the level of costimulation is important for the generation of anti-tumor immunity and for host survival. In addition, tumors appear to be able to evade the immune response by downregulating the expression of B7-2 below a threshold level.     

Modified vaccinia virus ankara recombinants are as potent as vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses

Cancer vaccine regimens use various strategies to enhance immune responses to specific tumor-associated antigens (TAAs), including the increasing use of recombinant poxviruses [vaccinia (rV) and fowlpox (rF)] for delivery of the TAA to the immune system. However, the use of replication competent vectors with the potential of adverse reactions have made attenuation a priority for next-generation vaccine strategies. Modified vaccinia Ankara (MVA) is a replication defective form of vaccinia virus. Here, we investigated the use of MVA encoding a tumor antigen gene, carcinoembryonic antigen (CEA), in addition to multiple costimulatory molecules (B7-1, intercellular adhesion molecule-1, and lymphocyte function-associated antigen-3 designated TRICOM). Vaccination of mice with MVA-CEA/TRICOM induced potent CD4+ and CD8+ T-cell responses specific for CEA. MVA-CEA/TRICOM could be administered twice in vaccinia na?ve mice and only a single time in vaccinia-immune mice before being inhibited by antivector-immune responses. The use of MVA-CEA/TRICOM in a diversified prime and boost vaccine regimen with rF-CEA/TRICOM, however, induced significantly greater levels of both CD4+ and CD8+ T-cell responses specific for CEA than that seen with rV-CEA/TRICOM prime and rF-CEA/TRICOM boost. In a self-antigen tumor model, the diversified MVA-CEA/TRICOM/rF-CEA/ TRICOM vaccination regimen resulted in a significant therapeutic antitumor response as measured by increased survival, when compared with the diversified prime and boost regimen, rV-CEA/TRICOM/rF-CEA/TRICOM. The studies reported here demonstrate that MVA, when used as a prime in a diversified vaccination, is clearly comparable with the regimen using the recombinant vaccinia in both the induction of cellular immune responses specific for the "self"-TAA transgene and in antitumor activity.     

Therapeutic vaccination against murine lymphoma by intratumoral injection of recombinant fowlpox virus encoding CD40 ligand

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 on antigen-presenting cells is essential for the initiation of cell-mediated and humoral immune responses. Malignant B cells also express CD40 and respond to CD40L by enhancing expression of costimulatory molecules. In this study, we investigated the therapeutic antitumor effect of intratumoral administration of recombinant fowlpox virus encoding murine CD40L (rF-mCD40L) in a murine B-cell lymphoma model. BALB/c mice with established s.c. and widely metastatic A20 lymphoma tumors were treated with intratumoral injections of rF-mCD40L together with systemic chemotherapy. This combined chemoimmunotherapy resulted in complete tumor regression and long-term survival of the mice. Some tumor cells in the injected sites expressed the CD40L transgene and had increased expression of the CD80 and CD86 costimulatory molecules. The therapeutic effect was dependent on CD8 but not on CD4 T cells. Moreover, there was a requirement that the recombinant CD40L virus be injected directly into the tumor, as opposed to peritumoral or distant sites. Thus, rF-mCD40L injected directly into the tumor microenvironment enhances the immunogenicity of tumor B cells. The results support future plans for intratumoral injection of rF-mCD40L in patients with lymphoma.     

Induction of therapeutic T-cell immunity by tumor targeting with soluble recombinant B7-immunoglobulin costimulatory molecules.

Tumor targeting with immunomodulatory molecules is an attractive strategy to enhance the host's antitumor response. Expression of CD80 (B7-1) and CD86 (B7-2) costimulatory molecules in tumor cells has proven to be an efficient way to enhance their immunogenicity. Here, we studied the effects of tumor targeting with biotinylated recombinant soluble B7-1- and B7-2 immunoglobulin G molecules (bio-B7-IgG) using a pretargeting approach based on the sequential use of a biotinylated antitumor monoclonal antibody and avidin. Mouse RMA T-lymphoma cells bearing either bio-B7-1-IgG or bio-B7-2-IgG on their surface prime in vitro naive CD8+ CTLs, which are highly effective in adoptive immunotherapy, and induce therapeutic immunity when injected in tumor-bearing animals. In vivo targeting of established RMA tumors with bio-B7-IgG either cures tumor-bearing mice or significantly prolongs their survival. The antitumor response induced by targeted bio-B7-IgG depends on both CD4+ and CD8+ T cells. Moreover, tumor targeting with bio-B7-IgG in vivo is critical for both expansion in lymphoid organs and mobilization into the tumor of tumor-specific CD8+ CTLs. When targeting is performed on poorly immunogenic TS/A mammary adenocarcinoma, only bio-B7-1-IgG primes naive CTLs in vitro and cures or significantly prolongs the survival of tumor-bearing mice in vivo, confirming that the two costimulatory molecules are not redundant with this tumor. Altogether, these data suggest that tumor avidination and targeting with soluble bio-B7-IgG may represent a promising strategy to enhance the antitumor response in the host.     

Adjuvant GM-CSF cytokine gene therapy for breast cancer]

The aim of this study was to examine the enhancement of antitumor immunity of irradiated granulocyte macrophage-colony-stimulating factor (GM-CSF) gene-transduced mouse breast cancer cells. METHODS: BALBMC mouse were vaccinated subcutaneously with saline or irradiated mouse breast cancer cells, BALBMC (1 x 10(6)/mouse), infected or not infected with recombinant adenovirus harboring GM-CSF gene on day-7. Mice were injected with parental cells (1 x 10(5)/mouse) on day 0. RESULTS: No mice vaccinated with irradiated GM-CSF producing BALBMC cells developed a tumor during the observation period of up to 16 weeks, whereas 100% of mice injected with saline developed a tumor. CONCLUSION: Our study demonstrates the feasibility of this immunotherapeutic approach as a novel adjuvant cancer therapy after surgery for breast cancer.     

Autologous lymphocyte responses to adenovirus-B7-1-transduced human cancer cells.

Transfection of the costimulatory molecule B7-1 (CD80) into murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this study was to test autologous lymphocyte responses against freshly resected human cancers infected in vitro with an adenovirus vector expressing the B7-1 molecule (AdB7). Resected tumors (sarcomas, adenocarcinomas, melanomas, and multiple myelomas) were disaggregated into single-cell suspensions and divided into three groups: (a) native, noninfected tumor cells (TM); (b) AdB7-infected, B7-1(+) tumor cells (TMB7); and (c) control Addl70.3-infected, B7-1(-) tumor cells (TMAd). B7-1 expression was verified by flow cytometry. Autologous peripheral blood lymphocytes from these patients were tested for proliferative and cytotoxic activity against the three tumor groups. There was an increased lymphocyte-proliferative response against B7-1(+) tumor cells, particularly in the presence of interleukin-12 (IL-12) or low-dose IL-2. B7-1(+) tumor cells were also killed more efficiently than B7-1(-) tumor cells in natural killer cell-mediated cytotoxicity assays, and this was most significant when lymphocytes had been pretreated with IL-12. Human natural killer cells were found to express CD28, a receptor for B7-1. The high efficiency of AdB7-mediated gene transfer and the augmented B7-1-mediated lymphocyte responses suggest that AdB7 vectors may be effective in human cancer immunotherapy.     

Induction of effective antitumor immunity in a mouse brain tumor model using B7-1 (CD80) and intercellular adhesive molecule 1 (ICAM-1; CD54) transfection and recombinant interleukin 12.

Although tumor-specific T lymphocytes recognize tumor-associated antigens (TAA) present on their cell surface via major histocompatibility complex (MHC) molecules, T cells require other activating signals. These are provided by costimulatory molecules, including B7-1 (CD80), B7-2 (CD86) and intercellular adhesive molecule 1 (ICAM-1; CD54). Transfecting mouse tumor cell lines with the B7 gene can lead to primary tumor rejection and the establishment of protective immunity. However, some studies have shown that the B7 effect upon T-cell-dependent tumor immunity is limited. Therefore, we examined the antitumor effects of recombinant interleukin 12 (IL-12) and genetically engineered glioma cells expressing B7-1 or both B7-1 and ICAM-1. Vaccination of mice with B7-1-expressing tumor cells substantially inhibited the growth of subcutaneously inoculated gliomas but not those located in the brain. Vaccination with B7-1-expressing tumor cells and systemic recombinant IL-12 (rIL-12) was more effective than either B7-1-expressing tumor cells or rIL-12 alone. Our murine brain tumor model also showed that vaccination with tumor cells expressing both B7-1 and ICAM-1 combined with rIL-12 prolonged survival. We have demonstrated the therapeutic potential of vaccination with rIL-12 and tumor cells expressing both B7-1 and ICAM-1 in the control of glioma growth.     

Combination therapy of an orthotopic renal cell carcinoma model using intratumoral vector-mediated costimulation and systemic interleukin-2.

PURPOSE: Interleukin (IL)-2 therapy is currently used for therapy of renal cell carcinoma (RCC). However, it is only effective in approximately 10% to 15% of patients, showing a need for additional therapies. We have previously described a replication-defective fowlpox vector encoding three costimulatory molecules (B7-1, ICAM-1, and LFA-3), designated rF-TRICOM. Here, we show that intratumoral administration of rF-TRICOM in an orthotopic RCC model effectively enhances tumor immunogenicity and reduces tumor burden in mice and the combination of rF-TRICOM and IL-2 is more effective than either therapy alone. EXPERIMENTAL DESIGN: RCC cells were implanted under the capsule of the kidney, and mice were given rF-TRICOM intratumorally 14 days later. We compared the effect of rF-TRICOM, rF-granulocyte macrophage colony-stimulating factor (GM-CSF), and two doses of IL-2 and combinations of the above on antitumor efficacy and survival. Host CD4(+) and CD8(+) T-cell responses were also evaluated. RESULTS: The results show that (a) systemic IL-2 therapy was moderately effective in the reduction of tumor burden in an orthotopic RCC model; (b) a single intratumoral injection of rF-TRICOM and rF-GM-CSF significantly reduced tumor burden; (c) the addition of systemic IL-2 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in further reduction of tumor burden, decrease in the incidence of metastasis, and extended survival in tumor-bearing mice above that seen with either treatment alone; and (d) CD8(+) T cells played a critical role in the antitumor effect seen with rF-TRICOM/rF-GM-CSF + IL-2 therapy. Finally, the addition of systemic recombinant IL-15 or intratumoral vector-delivered IL-15 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in substantially more tumor-free mice than either therapy alone. CONCLUSIONS: These studies show that intratumoral administration of rF-TRICOM admixed with rF-GM-CSF is effective at reducing tumor burden in mice and the addition of IL-2 further contributes to this effect. These studies thus form the rationale for combination immunotherapy clinical trials in patients with RCC.