Idiotype Protein Research Articles

Dendritic Cell Vaccines Are Superior to Idiotype-KLH Protein Vaccines at Priming a Therapeutic, Tumor-Specific Immunity Against Multiple Myeloma.

Idiotype (Id) protein secreted by myeloma cells is the best-characterized tumor-specific antigen and has been used in clinical vaccination trials administered either as Id-keyhole limpet hemocyanin (KLH) protein vaccines or as Id-pulsed dendritic cell (DC) vaccines. In this study, we used the 5TGM1 myeloma murine model to compare the efficacy of Id-KLH (protein vaccines) versus DCs pulsed with Id-KLH (DC vaccines), two formulas commonly used in clinical trials, to induce tumor-specific immunity, to protect mice from developing myeloma, and to treat myeloma-bearing mice. Vaccinations consisted of three weekly, subcutaneous injections of the protein conjugates (100 μg/injection) or Id-KLH-pulsed, bone marrow-derived mature DCs (106 cells/injection). Following each vaccination, GM-CSF (200 ng/day/mouse) was injected subcutaneously (adjacent to the vaccination sites) for three consecutive days. We found that, although the protein vaccines induced significantly higher titers of specific antibodies, DC vaccines were superior at inducing tumor-specific, cellular immune responses, evident by increased IFN-γ production, T cell proliferation, and cytotoxic T cell activities against the tumor cells. As prophylactic treatments, protein and DC vaccines were equally efficient at protecting mice from subsequent tumor challenge. However, only DC vaccines induced therapeutic immunity in tumor-bearing mice: DC vaccinations not only retarded tumor growth but also eradicated established tumors in more than 50% of mice, which was associated with an induction of potent, tumor-specific type-1 (IFN-γ) and type-2 (IL-4) T-cell responses. DC-vaccinated mice also showed a more pronounced increase in the percentages of CD8+ T cells in their spleens after in vitro stimulation with irradiated myeloma cells and increased tumor-specific cytotoxicity in enriched CD8+ T cells over the whole splenic cells, suggesting that CD8+ T cells play a major role in killing tumor cells in vivo. Furthermore, surviving mice were also protected from rechallenge with the myeloma cells, indicating that memory T cells existed and were functional. Thus, our results show that Id-based DC vaccines may be a favorable vaccine for immunotherapy in MM. Further studies are required to optimize DC vaccination protocols to achieve therapeutic efficacy in clinical trials.

Cloning of Idiotype Immunoglobulin Genes in B Cell Lymphomas by Anchored PCR and Production of Individual Recombinant Idiotype Vaccines in E. coli.

Abstract The hypervariable regions (idiotypes) of the immunoglobulins (Ig) expressed by lymphoma cells represent individual and specific tumor antigens. Purified or recombinant lymphoma-derived idiotype (Id) may be formulated into an individual vaccine and administered to lymphoma patients to elicit a therapeutic tumor-specific host immune response. We have developed an expression strategy based on anchored PCR amplification of lymphoma-derived cDNA. The Id variable (V) gene segments are inserted into a prokaryotic expression vector, and recombinant Fab fragment corresponding to the native Id protein is expressed in E.coli. This approach was applied to 106 biopsies with confirmed lymphoma involvement (47 follicular, 14 mantle cell, 6 diffuse large cell NHL, 22 myelomas, 6 CLL, and 6 other NHL types) and 8 non-lymphomatous control samples with the aim to investigate the feasibility of this production strategy in a large cohort of NHL patients, and to assess the yield and purity of the recombinant Id vaccines under large-scale production. According to clonality criteria defined to minimize the likelihood of mistakenly interpreting occasional repeat sequences (observed with low frequency in the control biopsies) as lymphoma-derived Id, cloning of Id VH and VL segments was successful in 95% of lymphoma biopsies. Expression of recombinant Fab fragment was successful (> 1 mg) in 93% of lymphomas. Fab yield was variable and ranged from 1.2 to 250 mg, whereas Fab purity was >95% in 95% of the cases after affinity chromatography purification. One Fab preparation only was contaminated with E.coli protein by western blot. MM had a trend towards superior Fab yields but this finding may merely reflect a selection for cases with secreted paraprotein, i.e. Ids with favorable folding properties and solubility. No other NHL type was associated with inferior expression levels. Fabs with lambda-type light chain had a trend towards lesser yield (p=0.0016) and purity (p=0.029) than kappa-Fabs. As expected, 25 of 38 follicular lymphoma cases had acquired novel potential glycosylation sites within their Id V segments (preferentially in CDR regions), whereas 34 of 40 other lymphomas lacked such sites (p<0.0001). Nevertheless, such sites did not influence the Fab yield (p=0.97). All other structural Id features (isotype, V gene usage, hypermutation status, CDRIII charge) also had no adverse influence on Fab expression. In summary, A-PCR cloning of Ig transcripts has a high success rate for the identification and analysis of Id genes in all major types of B-cell lymphoma. A-PCR with expression of idiotype protein in E. coli as a recombinant Fab fragment provides a solid basis for production of individual Id vaccines of all major IgH and IgL classes. The short culture time required for Id expression in bacteria in comparison to eukaryotic cell cultures may facilitate clinical trials and reduce production costs.

A Phase I Study of Immunization of Indolent Non Hodgkin's Lymphoma Patients with Autologous Monocyte-Derived Dendritic Cells Loaded with Heat Shocked and Killed Autologous Tumor Cells.

B-cell malignancies represent a potential target for anti-cancer vaccination programs due to the expression of tumor-specific antigens. Although immunization with tumor-derived idiotype protein is a frequently used procedure, vaccination with DCs loaded with killed tumor cells may activate response to a much wider range of antigens, without requiring prior molecular identification of such determinants. Furthermore, such DC-based vaccines could be available to all patients, irrespective of the HLA type. To evaluate the safety and tolerability of this approach, 18 patients with measurable relapse/refractory follicular (FCL; n= 12) and lymphoplasmocytoid (n= 6) lymphoma have been enrolled in a phase I study. Median prior number of treatment regimens was 2 (range 1–5) comprising 4 patients treated with high-dose chemotherapy supported by autologous stem cell transplantation. The vaccination was started after at least 6-months from the last chemotherapy treatment. All patients were evaluable for toxicity and 16/18 patients for efficacy with a median follow-up of 12.5 months (range 3–29 months). Each patient received 4 intradermal/subcutaneous injections at 2-weekly intervals of 50x10e6 tumor-loaded DCs. Immature DCs were generated by 5-days culture of autologous monocytes in the presence of IL-4 and GM-CSF. After selection by immunomagnetic technique, autologous CD19+ tumor cells, harvested from lymph nodes (n= 12) and/or peripheral blood (n= 6), were heat shocked and then irradiated by UVC. DCs were loaded for 48 hrs with killed tumor cells and then, to induce their maturation, were cultured for 12 hrs in the presence of TNF-alfa. Overall, vaccinations were well tolerated and no autoimmune reactions were observed. Mild erythema in the site of injection developed in the majority of patients (12/18), but only in 2 cases induration and extended erythema was observed. Six of 16 (37.5%) evaluable patients had objective responses. Two patients had partial responses (PR). One is still in PR and the other had a PR lasting 7 months. Four patients had complete remission (CR). Two patients are still in CR and the other 2 patients had a mean CR duration of 14.5 months. The remaining 10 patients had stable disease (n=5) or progressive disease (n=5). The overall monitoring of immune responses is ongoing. However, in one patient in PR, we evaluated the frequency of anti autologous tumor-specific T cells, by ELISPOT assay for IFN-gamma, on pathologic lymph nodes harvested before and after 2 months from the last vaccination. A significant increase of specific T-cell frequency was observed in the post-vaccination lymph node, compared to the tissue sample taken before vaccination. Moreover, evaluation of CD8+ T cell maturation markers, by analysis for CCR7 and CD45RA expression, indicated a shift of tumor-infiltrating T cells towards memory and effector stages in the lymph-node isolated after vaccination. In conclusion, injection of DCs loaded with killed tumor cells is a well-tolerated procedure achieving clinical and immunological responses also in the presence of significant tumor burden. However, further strategies, following DC-vaccination, are needed to ensure durable immune and clinical responses.

Failure of Vaccination With Idiotypic Protein or DNA, (+/???IL-2), the Depletion of Regulatory T Cells, or the Blockade of CTLA-4 to Prolong Dormancy in Mice With BCL1 Lymphoma

Immunization of mice with the idiotype (Id) immunoglobulin from the murine B cell lymphoma, BCL1, before inoculating tumor cells can induce tumor dormancy. In this model, the tumor cells grow for a short period of time and then regress. The mice live for months or years with approximately 1 million tumor cells in their spleens. Some mice relapse due to decreases in the anti-Id antibody titers or the development of mutations in the residual tumor cells which render them refractory to negative signaling by the anti-Id antibody. In this study we determined whether we could eliminate the residual dormant cells by using a DNA vaccine against the Id or by immunomodulation of T-cell subsets in vivo. Our results demonstrate that dormancy can be maintained by further immunizations with either the BCL1 Id protein or DNA vaccine encoding its single-chain Fv fragment. We also found that a cytotoxic T-cell response was not induced by either in vivo administration of vaccine alone or by the vaccine plus interleukin-2. In addition the injection of anti-cytotoxic T-lymphocyte-associate antigen did not prolong dormancy. Finally, the in vivo administration of anti-CD25 to deplete regulatory T cells did not prolong dormancy. Dormancy in this model is dependent primarily upon anti-Id antibodies, our results suggest that other strategies to target residual dormant BCL1 cells are warranted. They also suggest that the elimination of dormant tumor may represent a greater challenge than the elimination of primary tumors.

Current status of therapeutic vaccines for non-Hodgkin??s lymphoma

Therapeutic vaccines targeting B cell lymphoma idiotype have reached an advanced stage of clinical development, with three multicenter randomized clinical trials ongoing. This review describes the rationale and development of this immunotherapeutic approach, the design of current phase III trials, and other active vaccination approaches likely to move forward into clinical testing for lymphomas. Several groups have achieved promising results in phase II trials of patient-specific idiotype vaccines, with very few side effects noted. Anti-idiotype antibodies, in addition to cytotoxic T cells, are now believed to be important effectors of antitumor immunity after idiotype vaccination. The manufacturing of autologous tumor idiotype proteins is being rapidly refined by the use of molecular technologies. Two trials involving more than 1000 patients are now under way, which use idiotype vaccination after induction chemotherapy; one trial completed accrual in early 2004. A third trial opened in 2004, using rituximab followed by idiotype vaccine with maintenance booster vaccines continuing throughout the period of normal B cell recovery. In accordance with the United States Food and Drug Administration, progression-free survival serves as the accepted primary efficacy endpoint in these studies. Lymphoma idiotype vaccination represents a promising immunotherapeutic approach targeting a patient-specific tumor antigen. The results of pivotal phase III trials for three first-generation idiotype vaccines will become available in the next several years. Advanced manufacturing techniques should permit application of this tailor-made treatment to large numbers of non-Hodgkin's lymphoma patients.

Toward Personalized Immunotherapy for Non-Hodgkin Lymphoma

The idiotypic determinants of B-cell lymphomas, formed by cell-specific rearrangement of the immunoglobulin genes, are unique and are therefore a suitable target against which to direct immunotherapy. Recent advances in our understanding of the fundamental mechanisms behind an effective immune response, coupled with advances in genetic engineering techniques, have led to a renewed interest in immunotherapy. Early clinical studies have confirmed the immunogenicity of the idiotypic antigen in patients with lymphoma. This review discusses the different methods of idiotypic vaccination currently under investigation in the clinic, including protein, genetic, and cellular vaccines. Protein vaccines are the most clinically advanced, with phase III trials of idiotypic protein linked to GM-CSF currently underway. DNA vaccines are easier to produce but to date only appear to be weakly immunogenic in man. Dendritic cell vaccines have shown promise but their use may be limited by the complexity of this approach. This review also highlights other approaches not yet in the clinic but that have shown promise in the laboratory, such as viral vaccines and T-cell therapy.

A Novel Method for Producing Custom-Made Idiotype Vaccines for Lymphoma Immunotherapy Using a Cell-Free Expression System.

Idiotype vaccines have been used as a customized immunotherapy for lymphoma. This strategy takes advantage of a unique antigen (either immunoglobulin or T cell receptor) found on the lymphoma cells of each patient. To make this personalized vaccine feasible for widespread use a production method, which is rapid and relatively inexpensive will be required. In an E. coli-based cell-free protein expression system we were able to make ScFv proteins derived from the immunoglobulins of a mouse B cell lymphoma, 38C13, in high yields. We have used this cell-free system to make 38C13 ScFv proteins fused to either the cytokine mGM-CSF or a 9 amino acid peptide derived from hIL-1b. The 9 amino acid fragment of hIL-1b was chosen because it is known to retain immunostimulatory function while lacking pyrogenicity. We were able consistently to produce these proteins in high yield and to purify them using a histadine tag at the terminus of the molecule. The ScFv portion of the molecule is folded properly, as assayed by its binding to an anti-idiotype Ab. Similarly, the mGM-CSF fused to the ScFv can be recognized by an anti-mGM-CSF Ab. The mGM-CSF portion is biologically active as determined by its ability to support the growth a mGM-CSF dependant cell line. We immunized C3H mice with either the ScFc-hIL-1b or the ScFv-mGM-CSF fusion protein and we were able to induce an idiotype-specific Ab response, which recognizes the native idiotype protein in ELISA assays as well as the 38C13 tumor as detected by flow cytometry. Most importantly, in the highly aggressive 38C13 lymphoma model system, three bi-weekly vaccinations with either fusion protein protected mice from tumor challenge with the syngeneic 38C13 tumor. These data establish that a cell-free protein expression system can work for producing individualized idiotype vaccines for lymphoma. (This work was supported by NIH grant CA34233)

Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes

The clonotypic surface Ig receptor expressed by malignant B cells, idiotype, is a tumor-specific antigen and an attractive target for active immunotherapy. While Ab’s specific for tumor idiotype have been well described in patients with B cell malignancies, the precise antigenic epitopes in human idiotype recognized by autologous T cells remain largely unknown. We report here that T cell lines generated from lymphoma patients actively immunized with idiotype protein specifically recognized multiple, unique immunodominant epitopes in autologous tumor idiotype. Synthetic peptides corresponding to hypervariable, but not framework, regions of Ig heavy chain specifically stimulated CD4+ and CD8+ T cells to proliferate and secrete proinflammatory cytokines in an MHC-associated manner. Detailed analysis revealed a minimal determinant of an immunodominant epitope, comprising critical residues at the amino terminus that may be a product of somatic hypermutation. Association of idiotype-specific T cell responses with previously documented molecular remissions in idiotype-vaccinated patients suggests that the newly identified T cell epitopes may be clinically relevant. Such antigenic epitopes may serve as candidates for novel peptide-vaccine strategies, and as tools to selectively expand tumor antigen–specific T cells for adoptive immunotherapy and for monitoring T cell immunity in vaccinated patients.

Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes.

The clonotypic surface Ig receptor expressed by malignant B cells, idiotype, is a tumor-specific antigen and an attractive target for active immunotherapy. While Ab's specific for tumor idiotype have been well described in patients with B cell malignancies, the precise antigenic epitopes in human idiotype recognized by autologous T cells remain largely unknown. We report here that T cell lines generated from lymphoma patients actively immunized with idiotype protein specifically recognized multiple, unique immunodominant epitopes in autologous tumor idiotype. Synthetic peptides corresponding to hypervariable, but not framework, regions of Ig heavy chain specifically stimulated CD4(+) and CD8(+) T cells to proliferate and secrete proinflammatory cytokines in an MHC-associated manner. Detailed analysis revealed a minimal determinant of an immunodominant epitope, comprising critical residues at the amino terminus that may be a product of somatic hypermutation. Association of idiotype-specific T cell responses with previously documented molecular remissions in idiotype-vaccinated patients suggests that the newly identified T cell epitopes may be clinically relevant. Such antigenic epitopes may serve as candidates for novel peptide-vaccine strategies, and as tools to selectively expand tumor antigen-specific T cells for adoptive immunotherapy and for monitoring T cell immunity in vaccinated patients.

Adoptive immunotherapy with antigen-specific T cells in myeloma: a model of tumor-specific donor lymphocyte infusion

Although partial remissions rates of up to 60% are obtained with conventional therapeutic regimens, multiple myeloma is essentially an incurable disease with a median survival of approximately 30 months. Allogeneic stem cell transplantation (SCT) results in a high percentage of complete remissions (CRs), but it can be associated with significant treatment-related mortality. Recent clinical studies have shown that highly immunosuppressive, yet nonmyeloablative, doses of fludarabine-based chemotherapy can result in alloengraftment. However, even with a reduction in treatment-related mortality, success with allogeneic SCT is limited by the significant risk of relapse. The goal of the strategy described is to transfer tumor antigen-specific immunity induced in the stem cell donor to the allogeneic SCT recipient to reduce relapse. Donors are immunized with a well-defined vaccine, specific for the patient’s tumor. The allogeneic SCT is performed with a conditioning regimen consisting of cyclophosphamide and fludarabine, and the stem cell source is blood mobilized with filgrastim, which could potentially enhance the transfer of a larger number of tumor-specific T cells in the allograft, as compared to bone marrow. Donor immunization with myeloma idiotype protein in the setting of a nonmyeloablative SCT may represent a novel strategy for the treatment of myeloma.

Improved immunogenicity of a self tumor antigen by covalent linkage to CD40 ligand.

The interaction between the CD40 ligand (CD40L) and CD40 on antigen-presenting cells (APCs) is critical in promoting humoral and cellular immune responses. Agonistic anti-CD40 monoclonal antibody and soluble CD40L can act as powerful adjuvants to promote vaccination, but usually require repeated high-dose treatment. In this study, we demonstrate that the adjuvant effect of CD40L can be greatly improved by directly linking the antigen to CD40L. We constructed a fusion protein (Id-CD40L) consisting of the extracellular domain of CD40L and the idiotype (Id) protein, a weakly immunogenic tumor-specific antigen derived from the murine 38C13 B-cell lymphoma. The soluble Id-CD40L fusion protein retained CD40 binding activity and stimulated CD80 and CD86 upregulation and interleukin (IL)-12 production by macrophages. Immunization of mice with Id-CD40L without adjuvants resulted in high titers of anti-Id Abs dominated by the IgG1 isotype and protected the mice from subsequent lethal tumor challenge. In a dose-response study, we demonstrated that Id-CD40L elicited anti-Id antibody (Ab) responses in all immunized animals, even at a dose as low as 0.5 microg. Immunization with free Id and an IgG-CD40L fusion protein, which was identical in structure to Id-CD40L but lost the Id determinant, resulted in significant lower anti-Id responses, indicating that physical linkage between the tumor antigen and CD40L was required for the optimal immune response. These results demonstrate that fusing CD40L to a candidate antigen can greatly improve the adjuvant activity of CD40L. This approach may be useful in developing vaccines for a variety of malignant and infectious diseases.

Immunotherapy for lymphomas.

A growing list of immunotherapeutic strategies is now being employed to combat lymphoid malignancies. These efforts are warranted given that B-cell lymphomas, particularly those of the common follicular subtype, are among the most "immune-responsive" of all human cancers. Although systemic cytokine therapies for B-cell malignancies have been largely disappointing to date, monoclonal antibody therapies, principally the anti-CD20 antibody rituximab, have already made enormous impact on the treatment algorithm for many B-cell lymphomas. Therapeutic vaccines targeting the tumor-specific immunoglobulin idiotype have demonstrated promising results against lymphomas in phase I/II studies and are currently being evaluated in phase III randomized trials. Additional vaccine therapies being developed include those based on dendritic cells, recombinant idiotype proteins, DNA, heat shock proteins, and gene-modified tumor cells. It is hoped that immunotherapeutic agents, used in tandem or in combination, may someday allow effective treatment of lymphoid malignancies and delay or even replace the need for conventional cytotoxic therapies.

CTLA4 blockade maximizes antitumor T-cell activation by dendritic cells presenting idiotype protein or opsonized anti-CD20 antibody-coated lymphoma cells.

CTLA4 is a negative regulator of the costimulatory signals induced by the interaction of CD28 on T cells and B7 on dendritic cells (DCs). Antibodies (Abs) against CTLA4 can block its function and increase the activation of T cells primed to recognize antigens. The effect of CTLA4 blockade on the cross-presentation of tumor antigens by DCs to T cells was examined. Immune T cells and DC precursors were collected from patients receiving idiotype protein-pulsed DC vaccines, exposed to antigen, and examined for antitumor activity by measuring intracellular cytokine production by FACS. Idiotype-specific activation occurred in CD8+ and CD4+ T-cell populations and was up to 58 fold higher with CTLA4 blockade. These T cells could be expanded quickly and maintained tumor cytolytic activity. T-cell responses to whole tumor cell-pulsed DCs were then examined. DCs contain Fc receptors and efficiently phagocytose lymphoma cells when coated with opsonizing anti-CD20 Abs. Within a few hours, DCs ingested tumor cells and labeled proteins were observed in the cytoplasm. When anti-CD20 Ab-coated tumor-pulsed DCs were used in combination with CTLA4 blockade, up to 15 fold higher activation of Id-specific CD8+ and 3 fold higher CD4+ T cells resulted. Thus, CTLA4 blockade can enhance the measurement of Ag-specific T-cell responses and the expansion of T cells for clinical studies. In addition, the combination of CTLA4 blockade and Ab targeting of tumor to DCs is an effective method for the cross-presentation of tumor cell antigens.

Tumor lysate–specific cytotoxic T lymphocytes in multiple myeloma: promising effector cells for immunotherapy

AbstractThe idiotype protein, secreted by myeloma plasma cells, is a tumor-specific but weak antigen. Idiotype-based immunotherapy has been explored in myeloma patients with disappointing results. It is conceivable that myeloma cells contain a multitude of tumor antigens that can more effectively stimulate antitumor T cells. To explore the possibility of using whole myeloma cells as a source of tumor antigens for immunotherapy, the current study was undertaken to generate and examine the function of myeloma-specific cytotoxic T lymphocytes (CTLs) by using dendritic cells (DCs) pulsed with myeloma cell lysates as stimulating cells. After repeated stimulation, specific CTL lines, containing CD4+ and CD8+ T cells, were generated from myeloma patients. Our results show that these T cells not only recognized and lysed autologous myeloma protein–pulsed DCs, they also killed autologous primary myeloma cells. Occasionally, CTLs responded to autologous idiotype–pulsed DCs and to allogeneic primary myeloma cells. No cytolytic activity, however, was detected against autologous lymphocytes including B cells, suggesting that the T cells acted specifically against myeloma cells. Cytotoxicity against target cells was major histocompatibility complex class 1 and, to a lesser extent, class 2 restricted and was dependent mainly on the perforin-mediated pathway. CTLs secreted predominantly interferon-γ and tumor necrosis factor-α on antigenic stimulation, indicating a type 1 T-cell subset. These findings represent the first demonstration that tumor cell lysate–primed CTLs kill only myeloma cells, not autologous lymphocytes. This provides a rationale for myeloma cell–based immunotherapy in multiple myeloma.

Genetic vaccination for the immunotherapy of B-cell malignancies.

Vaccination protocols based on targeting of the idiotype expressed on malignant B cells have so far provided encouraging results in clinical trials. The essential requirement to induce an immune response is the inclusion of carriers to overcome T-cell tolerance. Chemical cross-linking of idiotypic protein is so far the method of choice to induce protective responses in human studies. Meanwhile, a flurry of alternative strategies to simplify vaccine production is being tested in murine model. Thanks to the advance in antibody engineering the two relevant antigenic domains of the lymphoma immunoglobulin can be assembled into an appropriate format, genetically linked to molecules that act as immunological adjuvants and directly delivered as plasmid DNA. Upon immunization, rejection of tumor cells may depend on cellular or humoral mechanisms, whose relative importance has not been entirely estimated. We have recently analyzed the specificity of anti-idiotypic antibodies induced by DNA vaccination and characterised the elements contributing to optimal anti-idiotypic responses.

Recent advances in multiple myeloma immunotherapy

Multiple myeloma (MM) responds to, but is not cured by, chemotherapy and may therefore be amenable to tumor-specific immunization in the setting of minimal residual disease. The idiotype of the monoclonal immunoglobulin expressed by the tumor provides a clear tumor-specific antigen. Patients with follicular lymphoma have unequivocally established that idiotypic vaccination, administered when patients have minimal residual disease, has an antitumor effect and potential to improve the clinical outcome. This result and preclinical studies demonstrating that MM cells display idiotypic peptides on their surface in a form suitable for recognition and killing by host T cells, foster the application of idiotypic vaccination in MM. The current vaccine production involves idiotype protein purification for each patient followed by conjugation to exogenous, immunogenic carriers in order to break immunological tolerance. Furthermore, recent advances in molecular cloning and development of novel antigen delivery systems are making it possible to streamline the production of equally or more effective idiotypic vaccines. Particularly, DNA vaccines utilising genetic carriers to target idiotype on dendritic cells in vivo have proven successful in preclinical models. Additional candidate T cell antigens, such as MUC1, the cancer-testis antigens, and telomerase have been identified as potential targets for immunization. The possibility of using whole myeloma cells as a source of tumor antigens for immunotherapy is also being actively explored. Finally, clinical studies have begun in which dendritic cells are generated ex vivo, loaded with tumor antigen(s), and reinfused to immunize patients.

Dendritic cell vaccines in the treatment of multiple myeloma: advances and limitations.

Dendritic cells (DCs) are antigen-presenting cells that play a key role in the induction of cytotoxic T-lymphocytes. Adjuvant immunotherapy with antigen-loaded DCs represents an attractive anticancer strategy for multiple myeloma (MM). Autologous DCs loaded with idiotypic protein or other myeloma-associated antigen have been used in several clinical trials. Preclinical and first clinical experience have provided valuable insights in the mechanisms of cellular immunity, but few, if any, patients with MM benefited from such vaccination. Taken together, the data suggest that antitumor T-cell responses fail in MM because of a deregulated cytokine network, downregulation of costimulatory surface receptor expression, and changes in T-cell repertoire, enabling tumor cells to escape immune effectors by preventing the antitumor immune response. We discuss current clinical protocols for DC-based immunotherapy in MM and review some strategies that may increase the efficacy of DC vaccines.

The development of a bicistronic plasmid DNA vaccine for B-cell lymphoma

Tumor vaccines are a promising alternative to chemotherapy for the treatment of metastatic cancer. To be effective and safe, a therapeutic cancer vaccine should specifically target antigens expressed only on metastatic tumor cells. A vaccine directed against the unique surface immunoglobulin or idiotype expressed on non-Hodgkin’s B-cell lymphoma fulfills these criteria, as both primary and metastatic tumor cells express tumor specific immunoglobulins. Using the murine 38C13 B-cell lymphoma tumor as a model system, a plasmid DNA vaccine was designed to express a bicistronic mRNA encoding both the light and heavy tumor immunoglobulin (idiotype) proteins expressed on the surface of the 38C13 tumor. To increase the immunogenicity of the plasmid DNA vaccine, each of the murine variable domains (light and heavy) were fused to their respective human immunoglobulin constant domains. In addition, a eukaryotic expression cassette was constructed to effect both high-level expression of the mouse/human chimeric immunoglobulin, and to elicit a protective immune response in vivo. Unique Sfi I restriction sites were used for the rapid cloning of any tumor specific immunoglobulin idiotype domains and a series of plasmid constructs were made to test changes to the J domain and/or the human C domain to insert the Sfi I restriction sites. Such changes were found to have significant effects on both expression and immunogenicity. Vaccination of mice with prototype idiotype vaccines was found to generate a protective immune response to the 38C13 tumor. This study indicates that a novel bicistronic plasmid DNA-based vaccine can be used to develop a tumor specific vaccine against B-cell lymphoma.

Translational development of active immunotherapy for hematologic malignancies.

The B-cell tumor-derived Ig receptor may be considered a model tumor antigen for cancer vaccine development. However, as a non-immunogenic, self-antigen, it also must be first rendered immunogenic by chemical or genetic fusion to carriers which enable the induction of protective antitumor immunity in experimental tumor models. Our group has demonstrated that active immunization of human patients with idiotypic protein vaccines containing soluble GM-CSF elicited antigen specific CD8+ T cell responses and antitumor effects. An alternative strategy to develop vaccines is the genetic fusion of tumor idiotype-derived single chain antigen with a chemokine moiety. Administration of these vaccines as fusion proteins or naked DNA vaccines may allow more efficient targeting of antigen presenting cells in vivo. Potent antitumor immunity was elicited in mice which was dependent on the generation of specific antibodies and both CD4+ and CD8+ effector T-cells. We propose that chemokine fusion may represent a novel, general strategy for formulating existing or newly identified tumor and HIV antigens into vaccines for cancer and AIDS, respectively, which elicit potent CD8+ T-cell immunity.

Anti-idiotypic DNA vaccines for B-cell lymphoma therapy.

The idiotypic determinants expressed by immunoglobulin at the surface of malignant B-cells provide specific targets for vaccination strategies. However, as self-antigens they are poorly immunogenic and vaccines must include carriers to improve immune responses. Chemical cross-linking of purified idiotypic protein is so far the only method which has been employed in clinical trials while a number of second-generation vaccines have been developed in mouse models. These strategies are based on the use of recombinant DNA technology to create fusion proteins that contain the idiotype genetically linked to molecules that act as immunological adjuvants. Fusion proteins can be made available to the immune system by direct delivery of naked DNA with great advantages in terms of time and costs. The most relevant approaches are listed and discussed with particular emphasis to the mechanism by which different molecules exert their adjuvant effect. The role of cellular versus antibody mediated suppression of tumor growth following Id vaccination is examined by comparing the most recent data on the mechanism of tumor protection. In addition, an analysis of the specificity of anti-idiotypic antibodies induced by scFv-DNA vaccination as compared to protein immunization is provided.