Anti-idiotype antibody response after vaccination correlates with better overall survival in follicular lymphoma.

Previous studies demonstrated that vaccination-induced tumor-specific immune response is associated with superior clinical outcome in patients with follicular lymphoma. Here, we investigated whether this positive correlation extends to overall survival (OS). We analyzed 91 untreated patients who received CVP chemotherapy (cyclophosphamide, vincristine, and prednisone) followed by idiotype vaccination. Idiotype proteins were produced either by the hybridoma method or by expression of recombinant idiotype-encoding sequences in mammalian or plant-based expression systems. We found that achieving a complete response/complete response unconfirmed (CR/CRu) to CVP and making an anti-idiotype antibody are 2 independent factors that each correlated with longer OS at 10 years (89% vs 68% with or without a CR/CRu, P = .024; 90% vs 69% with or without tumor-specific antibody production; P = .027). In the subset of patients who received hybridoma-generated vaccines, we found that anti-idiotype production was even more highly associated with superior OS (P < .002); this was the case even in patients with a partial response (PR) to CVP (P < .001).

Generation of CD8+ T cell-mediated immunity against idiotype-negative lymphoma escapees.

We investigated the ability of CpG-oligodeoxynucleotide to generate an anti-tumor CD8+ T-cell immune response and to synergize with passive antibody therapy. For these studies, we generated an antibody against the idiotype on the A20 B-cell lymphoma line. This antibody caused the regression of established tumors, but ultimately the tumors relapsed. The escaping surface IgG-negative tumor cells were resistant to both antibody-dependent cellular cytotoxicity and signaling-induced cell death. Addition of intratumoral CpG to antibody therapy cured large established tumors and prevented the occurrence of tumor escapees. The failure of the combination therapy in mice deficient for CD8+ T cells demonstrates the critical role of CD8+ T cells in tumor eradication. When mice were inoculated with 2 tumors and treated systemically with antibody followed by intratumoral CpG in just one tumor, both tumors regressed, indicating that a systemic immune response was generated. Although antibody therapy can eliminate tumor cells bearing the target antigen, it frequently selects for antigen loss variants. However, when a poly-specific T-cell response was generated against the tumor by intratumoral CpG, even large established tumors were cured. Such an immune response can prevent the emergence of antibody selected tumor escapees and provide long-lasting tumor protection.

Immunogenicity of a plasmid DNA vaccine encoding chimeric idiotype in patients with B-cell lymphoma.

B-cell lymphomas express tumor-specific immunoglobulin, the variable regions of which [idiotype (Id)] can serve as a target for active immunotherapy. Promising results have been obtained in clinical studies of Id vaccination using Id proteins.However, Id protein is laborious and time-consuming to produce. DNA vaccination is an attractive alternative for delivering Id vaccines, because Id DNA can be rapidly isolated by PCR techniques. DNA coding for lymphoma Id can provide protective immunity in murine models. In the present study, we performed a Phase I/II clinical trial to study the safety and immunogenicity of naked DNA Id vaccines in 12 patients with follicular B-cell lymphoma. The DNA encoded a chimeric immunoglobulin molecule containing variable heavy and light chain immunoglobulin sequences derived from each patient's tumor, linked to the IgG2a and kappa mouse immunoglobulin (MsIg) heavy- and light-chain constant regions chains, respectively. Patients in remission after chemotherapy received three monthly i.m. injections of the DNA in three dose escalation cohorts of four patients each (200, 600, and 1800 micro g). After vaccination, 7 of 12 patients mounted either humoral (n = 4) or T-cell-proliferative (n = 4) responses to the MsIg component of the vaccine. In one patient, a T-cell response specific to autologous Id was also measured. Anti-Id antibodies were not detectable in any patient. A second series of vaccinations was then administered using a needle-free injection device (Biojector) to deliver 1800 micro g both i.m. and intradermally (i.d.); 9 of 12 patients had humoral (n = 6) and/or T-cell (n = 4) responses to MsIg. Six of 12 patients exhibited humoral and/or T-cell anti-Id responses; yet, these were cross-reactive with Id proteins from other patient's tumors. Subsequently, a third series of vaccinations was carried out using 500 micro g of human granulocyte-macrophage colony-stimulating factor DNA mixed with 1800 micro g of Id DNA. The proportion of patients responding to MsIg remained essentially unchanged (8 of 12), although humoral or T-cell responses were boosted in some cases. Throughout the study, no significant side effects or toxicities were observed. Despite the modest level of antitumor immune responses in this study, DNA vaccine technology retains potential advantages in developing anti-Id immunotherapies. Additional studies are warranted to optimize vaccine dose, routes of administration, vector designs, and prime-boost strategies. These results will help guide the design of such future DNA vaccine trials.

The oncoprotein LMO2 is expressed in normal germinal-center B cells and in human B-cell lymphomas.

We previously developed a multivariate model based on the RNA expression of 6 genes (LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2) that predicts survival in diffuse large B-cell lymphoma (DLBCL) patients. Since LMO2 emerged as the strongest predictor of superior outcome, we generated a monoclonal anti-LMO2 antibody in order to study its tissue expression pattern. Immunohistologic analysis of over 1200 normal and neoplastic tissue and cell lines showed that LMO2 protein is expressed as a nuclear marker in normal germinal-center (GC) B cells and GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, natural killer (NK), and plasma cell neoplasms and was absent from nonhematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6, and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2). Our results warrant inclusion of LMO2 in multivariate analyses to construct a clinically applicable immunohistologic algorithm for predicting survival in patients with DLBCL.

Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation.

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients.

Tumor-specific clonal immunoglobulin expressed by B-cell lymphomas (idiotype [Id]) can serve as a target for active immunotherapy. We have previously described the vaccination of 4 patients with follicular lymphoma using dendritic cells (DCs) pulsed with tumor-derived Id protein and now report on 35 patients treated using this approach. Among 10 initial patients with measurable lymphoma, 8 mounted T-cell proliferative anti-Id responses, and 4 had clinical responses--2 complete responses (CRs) (progression-free [PF] for 44 and 57 months after vaccination), 1 partial response (PR) (PF for 12 months), and 1 molecular response (PF for 75+ months). Subsequently, 25 additional patients were vaccinated after first chemotherapy, and 15 of 23 (65%) who completed the vaccination schedule mounted T-cell or humoral anti-Id responses. Induction of high-titer immunoglobulin G anti-Id antibodies required coupling of Id to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH). These antibodies could bind to and induce tyrosine phosphorylation in autologous tumor cells. Among 18 patients with residual tumor at the time of vaccination, 4 (22%) had tumor regression, and 16 of 23 patients (70%) remain without tumor progression at a median of 43 months after chemotherapy. Six patients with disease progression after primary DC vaccination received booster injections of Id-KLH protein, and tumor regression was observed in 3 of them (2 CRs and 1 PR). We conclude that Id-pulsed DC vaccination can induce T-cell and humoral anti-Id immune responses and durable tumor regression. Subsequent boosting with Id-KLH can lead to tumor regression despite apparent resistance to the primary DC vaccine.