Surmounting tumor-induced immune suppression by frequent vaccination or immunization in the absence of B cells

Tumor-induced immune suppression is one of the most difficult obstacles to the success of tumor immunotherapy. Here, we show that established tumors suppress CD8 T cell clonal expansion in vivo, which is normally observed in tumor-free mice upon antigen-specific glycoprotein (gp) 96-chaperone vaccination. Suppression of CD8 T-cell expansion by established tumors is independent of tumor-associated expression of the antigen that is recognized by the CD8-T-cell receptor. Vaccination of tumor-bearing mice is associated with increased cellular recruitment to the vaccine site compared with tumor-free mice. However, rejection of established, suppressive tumors required frequent (daily) gp96 vaccination. B cells are known to attenuate T helper cell-1 responses. We found that in B-cell deficient mice, tumor rejection of established tumors can be achieved by a single vaccination. Accordingly, in tumor-free B-cell deficient mice, cognate CD8 cytotoxic T lymphocyte clonal expansion is enhanced in response to gp96-chaperone vaccination. The data have implications for the study of tumor-induced immune suppression and for translation of tumor immunotherapy into the clinical setting. Frequent vaccination with cellular vaccines and concurrent B-cell depletion may greatly enhance the activity of anticancer vaccine therapy in patients.     

Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism

A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.     

B7-1与CD40L在淋巴瘤免疫治疗中的协同作用

为了研究B7-1与CD40L共刺激途径在淋巴瘤的免疫治疗中的作用，探讨治疗淋巴瘤疫苗有效的作用方式，将A20淋巴瘤细胞株接种在BALB／C小鼠身上以构建荷瘤小鼠模型，将B7-1与CD40L表达载体单独或联合导入肿瘤内，以PBS和空载体作为对照，观察肿瘤生长的情况；应用肿瘤病理切片和HE染色技术观察肿瘤组织形态及淋巴细胞浸润情况，采用CCK-8试剂盒检测荷瘤小鼠脾CTL杀伤效应。结果显示，瘤内注射B7-1或CD40L表达载体可导致肿瘤生长延缓或体积缩小，两者联合注射较对照组和单独注射组的肿瘤消退效应明显增强。肿瘤形态学观察表明，治疗小鼠肿瘤局部有炎性细胞浸润并伴有大面积的坏死，肿瘤局限化；CCK8试剂盒检测显示小鼠脾CTL杀伤能力增强。结论：B7-1与CD40L疫苗对淋巴瘤具有治疗效应，两者联合治疗的效果优于单一治疗。质粒载体瘤内注射可作为一种安全有效的肿瘤疫苗作用方式。     

The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy

Anti-CD20 antibody immunotherapy effectively treats non-Hodgkin's lymphoma and autoimmune disease. However, the cellular and molecular pathways for B cell depletion remain undefined because human mechanistic studies are limited. Proposed mechanisms include antibody-, effector cell-, and complement-dependent cytotoxicity, the disruption of CD20 signaling pathways, and the induction of apoptosis. To identify the mechanisms for B cell depletion in vivo, a new mouse model for anti-CD20 immunotherapy was developed using a panel of twelve mouse anti-mouse CD20 monoclonal antibodies representing all four immunoglobulin G isotypes. Anti-CD20 antibodies rapidly depleted the vast majority of circulating and tissue B cells in an isotype-restricted manner that was completely dependent on effector cell Fc receptor expression. B cell depletion used both FcgammaRI- and FcgammaRIII-dependent pathways, whereas B cells were not eliminated in FcR common gamma chain-deficient mice. Monocytes were the dominant effector cells for B cell depletion, with no demonstrable role for T or natural killer cells. Although most anti-CD20 antibodies activated complement in vitro, B cell depletion was completely effective in mice with genetic deficiencies in C3, C4, or C1q complement components. That the innate monocyte network depletes B cells through FcgammaR-dependent pathways during anti-CD20 immunotherapy has important clinical implications for anti-CD20 and other antibody-based therapies.     

Tumorigenicity and immunogenicity of murine tumor cells expressing an MHC class II molecule with a covalently bound antigenic peptide

The significance of CD4+ lymphocytes and major histocompatibility complex (MHC) class II-restricted antigens in antitumor immunity has been demonstrated in several animal models as well as in some human tumors. However, because of the lack of known class II-restricted antigens, the participation of CD4+ cells in antitumor responses has not been well characterized. Recent reports showed that class II proteins covalently linked to an antigenic peptide could be constructed and cells expressing these fusion proteins were recognized by specific TH cells. The aim of this study was to determine the effect of the expression of a class II-peptide construct on the tumorigenicity and immunogenicity of transfected murine tumor cells. We have constructed a gene for I-Ed beta chain covalently coupled to the I-Ed-restricted TH cell determinant of sperm whale myoglobin (SWM132-145). This class II fusion protein was recognized by a specific TH cell line on the surface of COS-7 cells or BALB/c sarcoma cells. The sarcoma cells expressing the MHC-peptide complex were rejected by immunocompetent BALB/c mice, and in vivo T-cell subset depletion experiments suggested the importance of CD4+ cells in the rejection. Moreover, splenocytes from mice immunized with tumor cells expressing the I-Ed-SWM complex showed specific peptide recognition in vitro. Such covalent MHC-peptide complexes could prove useful in studies on the role of CD4+ lymphocytes in antitumor immune responses, and also in designing new, more effective vaccine approaches to the immunotherapy of cancer, as class II-restricted tumor-associated antigens are identified for human cancers.     

Active antitumor immunity elicited by vaccine based on recombinant form of epidermal growth factor receptor

Active immunotherapy targeting epidermal growth factor receptor (EGFR) should be another attractive approach to the treatment of EGFR-positive tumors. To test this concept, the authors evaluated the potential immune responses and antitumor activities elicited by dendritic cells pulsed with recombinant ectodomain of mouse EGFR (DC-edMER). Spleen cells isolated from DC-edMER-vaccinated mice showed a high quantity of EGFR-specific antibody-producing cells. EGFR-reactive antibody in sera isolated from vaccinated mice was identified and shown to be effective against tumors in vitro and in vivo by adoptive transfer. DC-edMER vaccine also elicited cytotoxic T-lymphocyte responses that could mediate antitumor effects in vitro and adoptive transfer in vivo. In addition, EGFR-specific cytokines responses were elicited by DC-edMER vaccine. Immunization with DC-edMER resulted in tumor regression and prolonged survival in mice challenged with Lewis lung carcinomas and mammary cancer models. Depletion of CD4+ T lymphocytes could completely abrogate the antitumor activity and EGFR-specific antibody responses, whereas the depletion of CD8+ T lymphocytes showed partial abrogation of the antitumor activity but antibody was still detected. Furthermore, tumor-induced angiogenesis was suppressed in DC-edMER-vaccinated mice or mice treated with antibody adoptive transfer. Taken together, these findings suggest the antitumor immunity could be induced by DC-edMER, which may involve both humoral and cellular immunity, and may provide insight into the treatment of EGFR-positive tumors through the induction of active immunity against EGFR.     

Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen.

Antigen-specific cancer immunotherapy and antiangiogenesis have emerged as two attractive strategies for cancer treatment. An innovative approach that combines both mechanisms will likely generate the most potent antitumor effect. We tested this approach using calreticulin (CRT), which has demonstrated the ability to enhance MHC class I presentation and exhibit an antiangiogenic effect. We explored the linkage of CRT to a model tumor antigen, human papilloma virus type-16 (HPV-16) E7, for the development of a DNA vaccine. We found that C57BL/6 mice vaccinated intradermally with CRT/E7 DNA exhibited a dramatic increase in E7-specific CD8(+) T cell precursors and an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with wild-type E7 DNA or CRT DNA. Vaccination of CD4/CD8 double-depleted C57BL/6 mice and immunocompromised (BALB/c nu/nu) mice with CRT/E7 DNA or CRT DNA generated significant reduction of lung tumor nodules compared with wild-type E7 DNA, suggesting that antiangiogenesis may have contributed to the antitumor effect. Examination of microvessel density in lung tumor nodules and an in vivo angiogenesis assay further confirmed the antiangiogenic effect generated by CRT/E7 and CRT. Thus, cancer therapy using CRT linked to a tumor antigen holds promise for treating tumors by combining antigen-specific immunotherapy and antiangiogenesis.     

Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes

Recent studies have identified a unique population of CD4+CD25+ regulatory T cells that is crucial for the prevention of spontaneous autoimmune diseases. Further studies demonstrated that depletion of CD4+CD25+ T cells enhances immune responses to nonself antigens. Because immune responses to malignant tumors are weak and ineffective, depletion of regulatory T cells has been reported to result in tumor regression. In the current study, using the weakly immunogenic MCA205 sarcoma and the poorly immunogenic B16/BL6/D5 (D5) melanoma, depletion of CD4+CD25+ T cells by the administration of anti-CD25 monoclonal antibodies (mAb), PC61 induced some tumor growth retardation, but all mice eventually succumbed to tumors. In our laboratory, immunotherapy by the transfer of tumor-immune T cells has demonstrated potent antitumor effects. A reliable source of tumor-reactive T cells has been lymph nodes (LN) draining progressive tumors. Therapeutic effector T cells can be generated by in vitro activation of draining LN cells with anti-CD3 mAb followed by culture in interleukin-2. In this system, PC61 mAb depletion of CD4+CD25+ T cells before or on day 8 of tumor growth resulted in increased sensitization in the draining LN. The therapeutic efficacy of activated tumor-draining LN cells from mAb depleted mice increased approximately three fold while maintaining specificity when tested in adoptive immunotherapy of established pulmonary metastases. Specific interferon-gamma secretion by LN T cells from mice treated with PC61 mAb 1 day before tumor inoculation increased significantly. However, this increase was not demonstrated with LN T cells from mice treated on day 8 despite their enhanced therapeutic reactivities. Our results indicate that although the antitumor immunity enhanced by the depletion of CD4+CD25+ T cells is insufficient to eradicate tumors, it augments the sensitization of immune T cells in the draining LN, thus, facilitating adoptive immunotherapy.     

Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy.

A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.     

B7.1/NHS76: a new costimulator fusion protein for the immunotherapy of solid tumors

Tumor evasion from immune surveillance is due to the anergic status of tumor-infiltrating lymphocytes, especially T cells. Inappropriate or absent expression of costimulatory molecules such as B7.1 and B7.2 lead to anergy and apoptosis of tumor-infiltrating T cells. To reverse this situation, a tumor-targeted fusion protein, human B7.1/NHS76, was generated by molecular engineering, which retains both the costimulatory activity of B7.1 and the tumor-targeting ability of NHS76 antibody. NHS76 is a human tumor necrosis therapy monoclonal antibody derived from phage display, and is capable of binding intracellular antigens, which are accessible and abundant in necrotic regions of tumors. As human B7.1 can interact functionally with murine B7.1 counter-receptors, the immunotherapeutic potential of this fusion protein was tested in 3 mouse tumor models (Colon 26, RENCA, and MAD109), and animal studies showed a 35% to 55% reduction in tumor volume. To modulate the immune inhibitory microenvironment in tumors, naturally occurring CD4+ CD25+ Treg cells were depleted by cytotoxic CD4 or CD25 antibodies. Combination therapy with anti-Treg and B7.1/NHS76 produced complete regression of established tumors and was associated with increased effector T-cell infiltration in tumors. Rechallenge experiments performed 3 months after mice attained complete remission by combination therapy showed that immunologic memory was established by these treatments. These studies indicate that the targeting of B7.1 to necrotic areas of tumors, where both the release of tumor antigens and infiltrating lymphocytes are prevalent, may be a new approach for the immunotherapy of solid tumors. Our results also suggest that the manifestation of immune-inhibitory factors such as the presence of Treg cells at the tumor site and associated draining lymph nodes may be a major cause for immune system failure to eradicate solid tumors.     

Monoclonal antibodies directed against the T-cell activation molecule CD137 (interleukin-A or 4-1BB) block human lymphocyte-mediated suppression of tumor xenografts in severe combined immunodeficient mice

A monoclonal antibody specific for the human analog of the murine T-cell activation molecule 4-1BB was generated and is shown here to react selectively with activated human CD4+ and CD8+ T lymphocytes. Treatment of these T cells in a one-way mixed lymphocyte culture with the anti-h4-1BB antibody enhanced the cell proliferation of the allostimulated lymphocytes. Previous studies in the mouse have shown that treatment of tumor-bearing mice with antibodies to 4-1BB augments anti-tumor immunity that is mediated by both CD4+ and CD8+ T cells. The authors consider the possibility that a similar approach may be efficacious for human cancer immunotherapy. This question was addressed by evaluating the effect of an anti-h4-1BB monoclonal antibody on human lymphocyte-mediated suppression of a human tumor xenograft in SCID mice. Mice treated with a control antibody and co-injected with the tumor and peripheral blood lymphocytes exhibited a lymphocyte dose-dependent suppression of tumor growth. In mice treated with the anti-h4-1BB antibody, the lymphocyte-mediated tumor suppression was completely eliminated and tumors grew progressively (as was observed in mice inoculated with tumors without lymphocytes). This monoclonal antibody specific for anti-h4-1BB, which augments the proliferation of allostimulated cells in vitro, blocks T-cell anti-tumor activity in vivo. These results suggest that although 4-1BB plays a role in the human peripheral blood lymphocyte-mediated suppression of tumor growth, antibodies to this molecule on human cells fail to stimulate anti-tumor activity, as was observed in tumor-bearing mice treated with an antibody to murine 4-1BB.     

Engineering and characterization of a novel fusion protein incorporating B7.2 and an anti-ErbB-2 single-chain antibody fragment for the activation of Jurkat T cells

The provision of the T-cell costimulatory molecule B7 to tumor cells can be an effective way to trigger a tumor-specific cytolytic T-cell response. One way to provide B7 to tumor cells would be to couple an antitumor antibody directly to B7. Such a molecule should target tumors displaying antigen and provide the costimulatory signal to T cells, resulting in the initiation of an antitumor T-cell response. To this end, a fusion protein was designed that incorporates a single-chain antibody fragment (scFv) to erbB-2 (Her2/neu), an oncogene product overexpressed by 30% to 50% of breast carcinomas, and the ECD of B7-2 (CD86). This fusion protein, expressed and purified from Pichia pastoris, was shown to retain binding activity to both counter receptors, erbB-2 and CD28. The fusion protein was also shown to target erbB-2-positive tumor cells and to deliver a CD28-specific T-cell costimulatory signal. These results suggest that a fusion protein engineered to target tumor cells and signal T cells for activation may be an effective means of cancer immunotherapy. Further studies should be performed to characterize the fusion protein in erbB-2 tumor-bearing mice for in vivo tumor targeting, biodistribution, and efficacy.     

Anti-CD70 antibodies: a potential treatment for EBV+ CD70-expressing lymphomas

A monoclonal antibody (Rituximab) directed against the B-cell surface antigen, CD20, is increasingly used as a therapy for B-cell lymphomas. However, CD20 is expressed on all normal mature B cells and hence is not a specific tumor target. In contrast, CD70 is expressed on highly activated lymphocytes as well as on many B-cell and T-cell lymphomas but is not expressed on the great majority of B cells and T cells. In this report, we have explored the potential utility of anti-CD70 monoclonal antibodies for treatment of CD70+ EBV+ B-cell lymphomas. Using two Burkitt's lymphoma lines (Raji and Jijoye) that express surface CD70 and a CD70- Burkitt's lymphoma line (Akata), we show that two different monoclonal antibodies directed against human CD70 allow rabbit and human complement to kill EBV+ B cells in a CD70-dependent manner in vitro. In the absence of complement, neither anti-CD70 antibody induced in vitro killing of CD70+ cell lines. Importantly, i.p. injection of anti-CD70 antibodies also inhibited the growth of CD70+ Burkitt's lymphoma cells in severe combined immunodeficient mice but did not inhibit the growth of CD70- Burkitt's lymphoma cells. These results suggest that anti-CD70 antibodies may be useful for the treatment of CD70+ B-cell lymphomas.     

Monoclonal immunotherapy with human monoclonal antibody(CLN-IgG) in glioma patients

It is well recognized that malignant gliomas escape an immune response by hiding behind the blood-brain barrier and by producing proteins that suppress systemic immunity. However, if gliomas can be made to be more immunogenic or if a tumor vaccine can be produced, then access to all tumor cells including those that infiltrate into the brain can be achieved through the patient's immune response. Several strategies have been investigated for immunotherapy. Laboratory studies and animal models have shown that these immune cells will attack the tumor cell, reduce the size of implanted tumors, and that the immune memory is sufficient to suppress tumor growth when the animal is rechallenges with a tumor implant. Since the development of hybridoma technology, monoclonal antibodies against human cancer cells have been produced and antigens have been identified. Hagiwara reported the production of a human monoclonal antibody, CLN-IgG, made by fusing UC 729-6, human lymphoblastoid B-cell line, with lymphocytes obtained from a patient with the cervical carcinoma. It has been reported that CLN-IgG recognized the antigen expressed in various histological types of human cancers including malignant gliomas. The effect of human monoclonal antibody(CLN-IgG) on malignant brain tumors was evaluated in patients with malignant glioma. Early phase II study was concluded that this specific immunotherapy with CLN-IgG is safe and effective therapy in patients with malignant glioma. We treated 10 cases of malignant gliomas with CLN-IgG. All patients had received radiotherapy and chemotherapy before this immunotherapy using the human monoclonal antibody. The human monoclonal antibody(CLN-IgG) was administered intravenously once or twice/week during 24 weeks. Six cases of glioblastoma, 1 medulloblastoma and 3 cases of potine glioma histologically unverified, were treated. Five cases of 6 glioblastomas died 4 to 12 months after this treatment, 3 cases of pontine glioma showed good responses, 2 cases showed marked decrease of tumor size and 1 case showed no regrowth of tumor on MRI imaging. For the above reasons, Human monoclonal antibody(CLN-IgG) might be useful as an immunotherapy of malignant gliomas.     

Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines

DNA vaccines are an appealing strategy for inducing cytotoxic T-lymphocyte and antibody responses against tumor cells as well as infectious agents. Dendritic cells (DCs) play a critical role in inducing immune responses, but their potential is not fully utilized in the DNA vaccine setting since they take up only a minor fraction of the injected DNA. Here we describe a novel DNA vaccination strategy based on the targeting of a modified tumor-associated antigen, the human papilloma virus (HPV) type 16 E7 protein, to DCs by a heat-shock protein (HSP) to enhance antigen presentation and immune responses. Specifically, a chimerical HPV-E7 and HSP70 fusion gene preceded with a leader sequence was constructed. When mice were immunized with this construct, the DNA is taken up by various types of cells, which then produce and secrete an HPV-E7-HSP70 fusion protein that is targeted to DCs by the HSP70 portion of the chimerical molecule for antigen presentation. In studies to test the efficacy of this strategy, we demonstrated that DNA vaccination with this secretory HPV-E7-HSP70 construct strongly enhanced an antigen-specific CD8+ T-cell response as well as a specific B-cell response in mice. Furthermore, this immunization approach not only protected mice against lethal challenge with an HPV E7-expressing tumor line (TC-1), but also showed a therapeutic effect against established tumors. The results of this study indicate that secretory HSPs can be broadly used to target tumor-associated antigens to DCs to enhance antigen-specific immune responses.     

Chemotherapy enhances CD8(+) T cell-mediated antitumor immunity induced by vaccination with vaccinia virus.

The use of immunotherapy or chemotherapy alone is generally ineffective against well-established tumors. To overcome this intrinsic resistance against therapy for tumors, we have attempted to combine immunotherapy with chemotherapy. In this study, we tried to induce a rapid antitumor effect via chemoimmunotherapy using a vaccinia viral vaccine as an immunotherapeutic agent with anticancer agents including epigallocatechin-3-gallate (EGCG) and conventional anticancer drugs. Although a combination of vaccinia-mediated vaccination and chemotherapy led to a strong inhibition of tumor growth, monotherapy alone failed to completely cure tumors. In contrast, intravenous injection of cisplatin (CDDP) or cyclophosphamide (CTX) after vaccinia virus vaccination led to complete regression of the established tumors. Interestingly, anticancer drugs appear to augment the antitumor effect of the vaccinia virus-mediated immunotherapy. This effect is mainly associated with the enhanced tumor-specific CD8(+) T cell immune response induced by vaccinia virus, which was demonstrated by antibody depletion. However, anticancer drugs alone failed to induce a significant enhancement of the tumor-specific CD8(+) T cell immune response. Taken together, these results suggest that combining vaccinia virus-based immunotherapy with anticancer drugs is particularly effective against established tumors by increasing the tumor antigen-specific CD8(+) T cell immune response, which is primed by vaccinia virus-mediated vaccination.     

IL-6-encoding tumor antigen generates potent cancer immunotherapy through antigen processing and anti-apoptotic pathways.

A naked DNA vaccine delivered by gene gun into antigen-presenting cells (APCs) has emerged as an attractive strategy for antigen-specific cancer immunotherapy. However, APCs have a limited lifespan, hindering their long-term ability to prime antigen-specific T cells. Furthermore, the potency of DNA vaccines is limited by their inability to process and present antigens. Interleukin-6 (IL-6) could play a role in immunity and cell apoptosis. We explored how the DNA vaccine encodes IL-6 to a model tumor antigen, human papilloma virus type-16 (HPV-16) E7. Mice vaccinated with IL-6/E7 DNA exhibited dramatic increases in E7-specific T-cell immunities, anti-E7 antibody responses, and impressive anti-tumor effects against E7-expressing tumors. The in vitro results revealed that IL-6 enhances DNA vaccine potency through the major histocompatibility complex class I pathway via direct and cross-priming effects. In addition, the delivery of IL-6/E7 DNA prolonged the survival of transduced dendritic cells (DCs) in vivo. Our results indicated that the IL-6/E7 DNA vaccine combined the mechanisms of enhancing antigen processing and presentation with prolonging the survival of DCs. Using IL-6 represents an innovative approach to enhancing DNA vaccine potency and holds promise for cancer prevention and immunotherapy.     

Anti-tumor immunity induced by in vivo adenovirus vector-mediated expression of CD40 ligand in tumor cells.

CD40 ligand (CD40L), the ligand for CD40 on antigen-presenting cells, is essential for the initiation of antigen-specific T cell responses, an important component of the immune response to tumors. This study is based on the hypothesis that in vivo genetic modification of tumor cells to express CD40L will trigger CD40 on local antigen-presenting cells to present tumor antigen to the cellular immune systems, thus eliciting anti-tumor immunity to suppress growth of the tumor. To examine this concept, subcutaneous tumors of three different murine tumor models in two strains of mice were infected with a recombinant adenovirus (Ad) vector expressing murine CD40L (AdmCD40L). In the B16 (H-2b, melanoma) and CT26 (H-2d, colon cancer) murine models, injection of AdmCD40L into established subcutaneous tumors resulted in sustained tumor regression and tumor-free status in >60% of animals. Intratumoral injection of AdmCD40L also significantly suppressed the growth of established, weakly immunogenic Lewis lung carcinoma (H-2b) tumors, but to a lesser extent. Ex vivo AdmCD40L-transduced tumor cells implanted in syngeneic hosts induced significant antitumor response against preexisting identical tumors at a distant site. Both in vivo and in vitro AdmCD40L modification of tumors to express CD40L elicited tumor-specific cytolytic T lymphocytes responses, and the transfer of spleen cells from treated mice efficiently protected naive mice against a subsequent tumor challenge. These results support the concept that transduction of tumors with a recombinant CD40L adenovirus vector may be a useful strategy for cancer immunotherapy.     

Enhanced immunity by NeuEDhsp70 DNA vaccine Is needed to combat an aggressive spontaneous metastatic breast cancer.

The rapid growth and ruthless metastasis of tumors demand effective broad immune responses. Dendritic cells (DCs) are critical in developing tumor vaccines. Recent investigations have been focused on modifying tumor antigens to target DCs to induce immune responses efficiently in vivo. In this study, human hsp70 was fused to the extracellular domain of rat Her2/Neu (NeuEDhsp70) for enhancing anti-tumor immunity in aggressive breast tumor models. NeuEDhsp70-conditioned DCs produced significant IL-12p40 and effectively presented NeuED antigens to T cells in vitro. NeuEDhsp70 DNA vaccine induced enhanced Neu-specific antibody and IFN-gamma-producing cellular immune responses in vivo. Although NeuEDhsp70 and NeuED DNA vaccines elicited comparable therapeutic anti-tumor immunity in an aggressive primary breast tumor model, NeuEDhsp70 DNA vaccine significantly increased survival and reduced metastasis in an aggressive spontaneous metastatic breast tumor model. Results from animal experiments with depletion of immune cells or with deficiency of CD40 molecules indicate that T cells and CD40 molecules are critical in the anti-tumor immunity induced by NeuEDhsp70 DNA vaccine. These observations suggest that NeuEDhsp70 DNA vaccine is a promising reagent to induce potent anti-tumor immunity to an aggressive spontaneous metastatic breast tumor.     

Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice

Current therapies for non-Hodgkin lymphoma commonly include CD20 mAb to deplete tumor cells. However, the response is not durable in a substantial proportion of patients. Herein, we report our studies in mice testing the hypothesis that heterogeneity in endogenous tissue CD20+ B cell depletion influences in vivo lymphoma therapy. Using highly effective CD20 mAbs that efficiently deplete endogenous mature B cells and homologous CD20+ primary lymphoma cells through monocyte- and antibody-dependent mechanisms, we found that lymphoma depletion and survival were reduced when endogenous host B cells were not depleted, particularly a rare IL-10-producing B cell subset (B10 cells) known to regulate inflammation and autoimmunity. Even small numbers of adoptively transferred B10 cells dramatically suppressed CD20 mAb-mediated lymphoma depletion by inhibiting mAb-mediated monocyte activation and effector function through IL-10-dependent mechanisms. However, the activation of innate effector cells using a TLR3 agonist that did not activate B10 cells overcame the negative regulatory effects of endogenous B10 cells and enhanced lymphoma depletion during CD20 immunotherapy in vivo. Thus, we conclude that endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells able to inhibit lymphoma depletion by CD20 mAbs. Consequently, B10 cell removal could provide a way to optimize CD20 mAb-mediated clearance of malignant B cells in patients with non-Hodgkin lymphoma.