Sindbis virus replicon particles encoding calreticulin linked to a tumor antigen generate long-term tumor-specific immunity

Alphavirus vectors have emerged as a promising strategy for the development of cancer vaccines and gene therapy applications. In this study, we used the replication-defective vaccine vector SIN replicon particles from a new packaging cell line (PCL) to develop SIN replicon particles encoding calreticulin (CRT) linked to a model tumor antigen, human papillomavirus type 16 (HPV16) E7 protein. The linkage of CRT to E7 in SIN replicon particles resulted in a significant increase in E7-specific CD8(+) T-cell precursors and a strong antitumor effect against E7-expressing tumors in vaccinated mice. SINrep5-CRT/E7 replicon particles enhanced presentation of E7 through the major histocompatibility complex (MHC) class I pathway by infecting dendritic cells (DCs) directly and pulsing DCs with lysates of cells infected by SINrep5-CRT/E7 replicons. Vaccination of immunocompromised (BALB/c nu/nu) mice with SINrep5-CRT/E7 replicon particles also generated significant reduction of lung tumor nodules, suggesting that antiangiogenesis may contribute to the antitumor effect of SINrep5-CRT/E7 replicon particles. Furthermore, SINrep5-CRT/E7 replicon particles generated long-term in vivo tumor protection effects and antigen-specific memory immunities. We concluded that the CRT strategy used in the context of SIN replicon particles facilitated the generation of a highly effective vaccine for cancer prophylaxis and immunotherapy.     

Dendritic cell vaccination with xenogenic polypeptide hormone induces tumor rejection in neuroendocrine cancer

PURPOSE: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice). EXPERIMENTAL DESIGN: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid-modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin. RESULTS: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8(+) T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-gamma-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8(+) cells. Immunohistochemistry revealed strong CD8(+) tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (-74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group. CONCLUSION: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.     

Vaccination with ant-idiotype antibodies mimicking a renal cell carcinoma-associated antigen induces tumor immunity

We have previously isolated and characterized 6 different internal image mouse monoclonal anti-idiotype antibodies (Ab2) directed against the paratope of mouse monoclonal antibody G250 (MAbG250, Abl), which specifically reacts with human renal cell carcinoma (RCC). These Ab2s (NUH31,44,51, 71,82 and 91) demonstrated specificity for the combining site of AbI, and appeared to recognize 2 partly overlapping idiotopes on AbI. In this study, we further characterize the fine specificity of the Ab2, investigate whether the immunogenicity of Ab2 could be enhanced by conjugation to a carrier and investigate the anti-tumor efficacy of Ab3 sera in mice challenged with RCC. Immunization of animals with Ab2 conjugated to keyhole limpet hemocyanin as carrier protein resulted in a 2-fold increase in antigen-specific anti-anti-idiotype antibodies (Ab3) as compared with immunization using Ab2 alone. Specific reactivity was observed with antigen-positive cell lysates, and all Ab3 sera contained immunoglobulin resembling AbI (Abl), as shown by competitive AbI -antigen binding assays. Fine-specificity studies of Ab3 sera revealed that the Ab2s can be divided into 4 mutually exclusive groups, showing that the 6 Ab2s recognize 4 slightly different idiotopes in the Abl binding pocket. Treatment of RCC-challenged mice with Ab3 sera resulted in significant tumor growth inhibition and lower tumor take rates as compared with control groups. Ab3 sera obtained from NUH-91-immunized animals showed superior characteristics as compared to the other Ab3 sera: no tumors remained after 5 weeks of Ab3-NUH91 treatment. Our findings indicate that the Ab2 elicit powerful anti-tumor effects in immune-competent animals.     

Induction or elimination of tumor-specific immunity against a chemically-induced rat tumor using auto-anti-idiotypic immunity

DA rat sarcomas P, and P, were induced by dimethylbenz (a) anthracene. A tumor-specific immune response of DA rats against P₁-tumor cells could be demonstrated at the humoral and cellular level. DA anti-P₁ antibodies were purified on fixed Prtumor cells and used as autoimmunogen in DA rats for the production of antiidiotypic antibodies. Such antiidiotypic antibodies could be demonstrated by using a solid-phase radioimmunoassay and by their ability to induce secondary type of DA anti-P₁ responses in vitro. In addition, such antibodies were able to induce cytotoxic T lymphocytes capable of eliminating P₁-tumor cells but not control tumor cells. In some of the auto-immunized DA rats enhanced P₁-tumor growth could be observed, indicating that the anti-idiotypic immune response had led to a selective ablation of idiotypic, potential anti-P₁ reactive T cells.     

Fas ligand-expressing tumors induce tumor-specific protective immunity in the inoculated hosts but vaccination with the apoptotic tumors suppresses antitumor immunity

The interaction between Fas and Fas ligand (FasL) is involved in the apoptotic death of a number of cells including lymphocytes. Forced expression of FasL in tumors can induce apoptosis of infiltrating Fas-positive T cells; accordingly, tumors can survive in the milieu of systemic immune responses. However, FasL-expressing murine lung carcinoma (A11) and melanoma (B16) cells did not develop subcutaneous tumors and FasL-expressing A11 (A11/FasL) cells produced few spontaneous lung metastatic foci in syngeneic mice. The mice that rejected A11/FasL cells were resistant to subsequent challenge of parent A11 but not irrelevant B16 cells. Vaccination of mice with UV-treated A11/FasL, but not UV-treated A11 cells, however, augmented the growth rate of A11 but not B16 tumors, both of which were subsequently inoculated. The number of lung metastatic foci of A11 cells was also increased in the mice that received UV-treated A11/FasL but not UV-treated A11 cells. Intraperitoneal injection of UV-treated A11/FasL cells resulted in the production of larger amounts of immunosuppressive TGF-beta in peritoneal exudate than that of UV-treated A11 cells. Expression of the CD80 costimulatory molecule in tissues where UV-treated A11/FasL cells were inoculated was lower than the expression at an untreated A11/FasL-injected site. Our results indicated that apoptotic FasL-expressing tumor cells could impair host immune responses against the tumors, in contrast to potent antitumor immunity generated by viable FasL-expressing tumors.     

In situ analysis of tumor-specific CTL effector and memory responses elicited by tumor vaccination.

In this study tumor-specific CTL effector and memory responses were analyzed in normal mice, in tumor bearing mice and in animals treated by active specific immunotherapy (ASI) with an autologous virus modified tumor vaccine. In the well defined DBA/2 mouse lymphoma model ESb, the tumor specific CTL response requires interactions of four different cell types and recognition of MHC class I and class II restricted tumor antigens either on host antigen presenting cells (APCs) or on the tumor cells. The most effective in situ activation of syngeneic tumor specific CTL can be generated within no more than 9 days by primary immunization in the ear pinna and restimulation in the peritoneal cavity. Here we describe modulatory effects of either cytokines or of virus infection of tumor stimulator cells during restimulation in the peritoneal cavity on the CTL memory response. The in situ peritoneal effector cell (PEC) response was augmented when Newcastle Disease Virus (NDV) was used to infect tumor cells which expressed the correct tumor associated antigen of the memory response but not when a third party tumor cell was infected and admixed. A response could also be augmented by co-administration of interferons-alpha, -beta or IL-2 and by pre-treatment with low dose cyclophosphamide. Therapeutic vaccination effects could be achieved in mice inoculated s.c. with this aggressive and metastatic tumor variant if the vaccine was given very early, i.e. 1-2 days after priming and when the vaccine was applied post-operatively, i.e. in situations of low tumor burden. A long-term CTL memory response could be demonstrated even two months after post-operative active specific immunotherapy with ESb-NDV vaccine.     

Tumor-associated antigen of spontaneous mammary tumor in rats

The present study was undertaken to detect the spontaneous mammary tumor-associated antigen ( MTAA ), and to find the cross-reacting antigen in chemically-induced mammary tumor. The antisera against spontaneous mammary tumor were raised in the WAF1 rats of the same strain and tested for the detection of tumor-associated soluble antigen of mammary tumor induced by N-ethylnitrosourea (ENU) and N-butylnitrosourea ( BNU ). The MTAA was found in the extract of spontaneous mammary tumor by the double immunodiffusion test, while it was not found in the extract of normal and fetal tissues, hyperplastic mammary gland, spontaneous fibroadenoma, and chemically-induced mammary tumor. On the other hand, the MTAA was not detected in the other types of tumors induced by ENU or BNU , i.e. gastric cancer, intestinal tumor, brain tumor, kidney tumor, bladder tumor, hemangioma, rhabdomyosarcoma, or leukemia. The spontaneous MTAA could not be detected in the spontaneous mammary tumor of C3H mice or human breast cancer either. The MTAA was extracted effectively by 3 M KC1. Furthermore, the MTAA was found in the cytoplasm of continuous established mammary tumor cell line ( SpMT -1) by the immunofluorescence test.     

Immunological tolerance and tumor rejection in embryo-aggregated chimeric mice – Lessons for tumor immunity

Background  

Rejection of transplanted tumors by the immune system is a rare event in syngeneic hosts, and is considered to be dependent on the local interaction of defensive immune reactions and tumor tolerance mechanisms. Here, we have enlisted the aid of a unique set of embryo-aggregated lineage chimeric mice derived from C57/BL6 and FVB donors to study the interplay between local and systemic tumor immunity and tolerance in rejection of mouse B16 melanoma cells, syngeneic to the C57/BL6 donor strain.     

Intratumoral vaccination with vaccinia-expressed tumor antigen and granulocyte macrophage colony-stimulating factor overcomes immunological ignorance to tumor antigen

Using a murine transitional cell carcinoma tumor model, MB49, which naturally expresses the male antigen HY, we evaluated whether tumor ignorance as determined by lack of a systemic immune response could be overcome by immunization with vaccinia expressed tumor antigen and granulocyte macrophage colony-stimulating factor. Systemic tumor ignorance of MB49 was demonstrated by the lack of a splenic HY-specific CTL response in MB49-bearing female mice. In contrast, we demonstrated HY-specific CTL priming in the draining lymph nodes. MB49-bearing female B6 mice were immunized with VVHY+VVGMCSF intratumorally or in the contralateral flank. Intratumoral VVHY, VVGMCSF, and keyhole limpet hemocyanin (to produce CD4 help) generated splenic HY-specific CD8 CTLs, whereas immunization with the combination in the contralateral flank or single agents given intratumorally failed to yield a splenic response. Purified male T cells injected intratumorally, as a source of HY antigen, also generated a HY-specific response, whereas contralateral immunizations did not. These finding expand the understanding of tumor immunological ignorance and support intratumoral vaccination as a strategy for immunotherapy of established tumors.     

Inoculation of human interleukin-17 gene-transfected Meth-A fibrosarcoma cells induces T cell-dependent tumor-specific immunity in mice

OBJECTIVE: The biological activities of interleukin-17 (IL-17), a newly cloned cytokine, have not been fully elucidated. The present study was designed to assess the in vitro and in vivo effect of transfecting the IL-17 gene into tumor cells. METHODS: A complementary DNA (cDNA) encoding human IL-17 (hIL-17) was obtained by polymerase chain reaction amplification from the human CD4+ T cell cDNA library and inserted into the plasmid pRc/cytomegalovirus to construct an expression vector for the hIL-17 gene. Murine Meth-A fibrosarcoma cells were transfected with the hIL-17 gene using the lipofectin method. The hIL-17 gene-expressing clone (Meth-A/IL-17) was selected and analyzed for cytokine expression by Northern blot. RESULTS: There was no significant difference in the in vitro proliferation rate among parent Meth-A, cells transfected with vector alone and Meth-A/IL-17 cells. When the tumor cells were transplanted subcutaneously into BALB/c nude (nu+/nu+) mice, there was no difference in in vivo growth rates among the three cell lines. Challenge with tumor cells in conventional BALB/c mice, however, resulted in the rejection of Meth-A/IL-17 cells, but the other two lines did grow. After immunization with Meth-A/IL-17 cells, the mice were rechallenged by parent Meth-A or syngeneic MOPC-104E plasmacytoma cells; the immunized mice rejected the Meth-A cells, but not the MOPC-104E cells. Injecting the anti-thy 1,2 (CD90), anti-CD4 or anti-CD8 monoclonal antibody into conventional BALB/c mice resulted in the resumption of in vivo growth of Meth-A/IL-17 cells, but injecting the anti-asialo GM1 antibody did not. Furthermore, flow cytometric analysis demonstrated a significant increase in the expression of major histocompatibility complex (MHC) class I and class II antigens and lymphocyte function-associated antigen-1 on Meth-A/IL-17 cells. CONCLUSION: Meth-A cells transfected with the hIL-17 gene can induce tumor-specific antitumor immunity by augmenting the expression of MHC class I and II antigens, and both CD4+ and CD8+ T cells may play important roles in inducing antitumor immunity, suggesting the possibility of developing a tumor vaccine incorporating IL-17-transfected tumor cells.     

Separation of the tumor rejection antigen of Rous sarcoma virus-induced murine fibrosarcoma

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was separated and some of its physicochemical properties were characterized. Cytosol and plasma membrane fractions were separated from Rous sarcoma virus (RSV)-induced CSA1M tumor cells. Immunization with membrane but not cytosol fraction of these tumor cells together with complete Freund's adjuvant resulted in complete protection against subsequent challenge with viable CSA1M cells. The TRA activity contained in the membrane fraction was recovered in the sodium dodecyl sulfate (SDS)-solubilized fraction after the SDS-extraction of CSA1M membranes. This CSA1M SDS-solubilized preparation gave protection against syngeneic RSV-induced CSA9F tumor cells as well as the homologous tumor cell type, but failed to induce resistance to RSV-unrelated tumor cells. The membrane or SDS-solubilized fraction from RSV-unrelated tumor cells was unable to generate anti-CSA1M protective immunity. Physicochemical analyses have demonstrated that TRA activity in the SDS-solubilized fraction was completely abolished by treatment with proteinase K but was only marginally affected after treatment with glycosidase mixture. When the SDS-solubilized preparation was applied to a Sephacryl S-300 superfine column, TRA activity was recovered in the range of molecular weight of 50-90 kD. Further fractionation of this TRA-positive fraction by SDS-polyacrylamide gel electrophoresis revealed that the molecular size of TRA is 56-68 kD. These results indicate that membrane proteins which were isolated from CSA1M tumor cells and have a molecular size of about 60 kD are capable of inducing RSV-induced tumor-specific in vivo protective immunity.     

Potent tumor-specific immunity induced by an in vivo heat shock protein-suicide gene-based tumor vaccine

Tumor cells harbor a repertoire of unique, mutated antigens and shared self-antigens but generally are incapable of provoking an effective immune response, likely because of inadequate antigen presentation by professional antigen-presenting cells. Heat shock proteins (HSPs) play important roles in eliciting innate and adaptive immunity by chaperoning peptides for antigen presentation and providing endogenous danger signaling. Although effective in inducing tumor-specific immunity in mice and in some clinical trials, tumor-derived HSPs have many limitations like vaccines, such as the technical difficulty of ex vivo preparation of adequate quantities of HSPs from the resected tumors of individual patients. Here we have developed an in vivo HSP-suicide gene tumor vaccine by generating a recombinant replication-defective adenovirus (Ad-HT) that coexpresses HSP70 and a herpes simplex virus thymidine kinase suicide gene. The combination of HSP70 overexpression in situ and tumor killing by thymidine kinase/ganciclovir treatment, but neither strategy alone, provoked potent systemic antitumor activities after intratumor injection of Ad-HT. Tumor-specific CD4+ and CD8+ T-cell responses were induced by Ad-HT intratumor injection. CD11c+ dendritic cells (DCs) isolated from mice treated with Ad-HT were able to prime tumor-specific CTLs. Collectively, these results indicate that the combination of tumor killing by activation of a suicide gene to release tumor antigens and in situ HSP70 overexpression to enhance DC antigen presentation overcomes host immune tolerance to tumor antigens, leading to the induction of potent antitumor immunity. Our findings may have broad relevance to the use of the in vivo HSP/suicide gene tumor vaccine in therapy for human solid tumors.     

ALK a novel lymphoma-associated tumor antigen for vaccination strategies

The discovery of Tumor Associated Antigens (TAAs) demonstrated that tumor cells can be specifically recognized by the immune system raising the hypothesis that tumors express antigens that Cytotoxic T Lymphocytes (CTLs) can potentially attack. The identification of immunogenic epitopes led to their use as targets to mediate the specific clearance of neoplastic cells by TAA targeting strategies such as vaccination strategies. One of the critical issues in the development of efficient vaccination protocols is the identification of the appropriate TAAs. The TAA should be effective as a "tumor rejection antigen" able to induce an immune response that will affect tumor growth. A distinct pathologic entity characterized by the expression of the Anaplastic Lymphoma Kinase (ALK) protein and named "ALKoma" has recently emerged within the heterogeneous group of CD30+ Anaplastic Large Cell Lymphoma (ALCL). ALK is a receptor tyrosine kinase whose expression is normally restricted to a few scattered cells in the nervous system. Its pathological expression in lymphoma cells is due to a chromosomal translocation that leads to the formation of an ALK-derived oncogenic fusion proteins. ALK fusion proteins ectopically over-expressed and constitutively activated in lymphoid cells play a key role in the neoplastic transformation by the aberrant phosphorilation of intracellular substrates that likely contributes to the molecular pathogenesis of ALCL. The high level of ALK expression in lymphoma cells and its direct role in lymphomagenesis, combined with the fact that normal ALK is expressed at low levels in the immune privileged nervous system, makes ALK an ideal lymphoma-specific target for immunotherapy of ALK+ALCL.