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The unique antigenic determinants (idiotype (Id)) of the immunoglobulin secreted by myeloma tumor can serve as a tumor-specific antigen for active immunotherapy. Our objective was to induce tumor-specific T-cell immunity in bone marrow transplant (BMT) donors to enhance antitumor effects of allografts. We vaccinated five HLA-matched sibling donors with myeloma Id proteins isolated from recipient plasma before bone marrow harvest. Recipients were administered booster Id immunizations following transplantation. Vaccination induced donor Id and carrier-specific cellular and/or humoral immune responses. Two recipients died within 30 days of BMT from transplant-related complications. Id and carrier-specific T-cell responses were detected in all three remaining patients post-, but not pre-BMT and persisted for 18 months. All three surviving patients converted from partial to complete responses following BMT. Two of the three patients remain disease-free 7 years and 8 years after BMT, and the third died of renal failure after 5.5 years while in complete remission from myeloma. Our results suggest that myeloma Id vaccination induces specific T-cell immunity in healthy donors which may be transferable by BMT, is associated with prolonged disease-free survival of recipients, and may represent a general strategy to enhance graft-versus-tumor effect in other malignancies for which defined tumor-specific antigens exist.

We translated these laboratory findings to a single-arm, phaseII clinical study in which patients with previously untreatedstage III/IV follicular lymphoma were initially treated intominimal residual disease (MRD) state with a uniformchemotherapy regimen, PACE (prednisone/doxorubicin/cyclophosphamide/etoposide), a modified ProMACE regimen(cyclophosphamide/etoposide/doxorubicin/mechlorethamine/procarbazine plus high-dose methotrexate with leucovorin rescueand prednisone) without methotrexate.

Lymphomas express a tumor-specific antigen which can be targeted by cancer vaccination. We evaluated the ability of a new idiotype protein vaccine formulation to eradicate residual t(14;18)+ lymphoma cells in 20 patients in a homogeneous, chemotherapy-induced first clinical complete remission. All 11 patients with detectable translocations in their primary tumors had cells from the malignant clone detectable in their blood by PCR both at diagnosis and after chemotherapy, despite being in complete remission. However, 8 of 11 patients converted to lacking cells in their blood from the malignant clone detectable by PCR after vaccination and sustained their molecular remissions. Tumor-specific cytotoxic CD8+ and CD4+ T cells were uniformly found (19 of 20 patients), whereas antibodies were detected, but apparently were not required for molecular remission. Vaccination was thus associated with clearance of residual tumor cells from blood and long-term disease-free survival. The demonstration of molecular remissions, analysis of cytotoxic T lymphocytes against autologous tumor targets, and addition of granulocyte-monocyte colony-stimulating factor to the vaccine formulation provide principles relevant to the design of future clinical trials of other cancer vaccines administered in a minimal residual disease setting.

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 μg/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 μg/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/μL but was able to speed the time of recovery of platelet counts to 100,000/μL (15v 20 days; P = .01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/μL by an average of at least 8 days as compared with cycle A-1 (without PIXY321;P ≤ .004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.

Purpose: Rituximab, a humanized monoclonal antibody directed to the CD20 antigen present on B lymphocytes, could potentially abrogate the humoral immune response to murine monoclonal antibodies or immunotoxins by depleting antibody-producing B cells. Experimental Design: A Phase II study of LMB-1, an immunotoxin targeting the Lewis Y tumor antigen, in combination with rituximab was conducted to test the hypothesis that rituximab could abolish or diminish the development of human antibodies to LMB-1. Five patients were treated in this study and received 375 mg/m2 rituximab on days 1 and 7 followed by 45 μg/kg/day LMB-1 on days 10, 12, and 14. The development of human antibodies against LMB-1 was detected using a serum neutralization and ELISA. Results: All five of the patients had a total suppression of circulating CD20/CD19 B-cell population before the administration of the first dose of the immunotoxin. Before rituximab treatment, the mean percentage of CD20/CD19-positive B cells in the five treated patients was 19.8% (range, 4.5–29.8%) of the total peripheral lymphocytes. After two doses of rituximab, CD20/CD19-positive B lymphocytes constituted ≤0.1% of the total peripheral lymphocytes. Despite absent circulating antibody-producing B cells, before and during LMB-1 treatment, all of the patients developed neutralizing antibodies to the immunotoxin by day 21 of drug administration, which prevented retreatment. Conclusions: Even though rituximab caused complete depletion of circulating CD20/CD19-positive B cells, it had no effect in suppressing the human antibody response to LMB-1 and may be of limited utility in suppressing human antibody responses to other immunogenic proteins.

Purpose: The idiotype (Id) of the immunoglobulin on a given B-cell malignancy is a clonal marker that can serve as a tumor-specific antigen. We developed a novel vaccine formulation by incorporating Id protein with liposomal lymphokine that was more potent than a prototype, carrier-conjugated Id protein vaccine in preclinical studies. In the present study, we evaluated the safety and immunogenicity of this vaccine in follicular lymphoma patients. Experimental Design: Ten patients with advanced-stage follicular lymphoma were treated with five doses of this second generation vaccine after chemotherapy-induced clinical remission. All patients were evaluated for cellular and humoral immune responses. Results: Autologous tumor and Id-specific type I cytokine responses were induced by vaccination in 10 and 9 patients, respectively. Antitumor immune responses were mediated by both CD4+ and CD8+ T cells, were human lymphocyte antigen class I and II associated, and persisted 18 months beyond the completion of vaccination. Specific anti-Id antibody responses were detected in four patients. After a median follow-up of 50 months, 6 of the 10 patients remain in continuous first complete remission. Conclusions: This first clinical report of a liposomal cancer vaccine demonstrates that liposomal delivery is safe, induces sustained tumor-specific CD4+ and CD8+ T-cell responses in lymphoma patients, and may serve as a model for vaccine development against other human cancers and infectious pathogens.

Malignant B-cells in Follicular Non-Hodgkin’s Lymphoma expresses a clonal idiotype immunoglobulin which can serve as the basis for a patient-specific anti-idiotype vaccine. In a previous single-arm Phase II study by Bendandi, et al (Nature Med5:1171–1177, 1999), we evaluated the ability of tumor-specific idiotype (Id) conjugated to keyhole limpet hemocyanin (KLH) administered concurrently with granulocyte-monocyte colony-stimulating factor (GM-CSF) adjuvant to induce complete remissions and molecular remissions in treated patients. The vaccine formulation induced a tumor-specific cytotoxic CD8+ and CD4+ T-cell response in patients in first complete remission after standard chemotherapy, as well as achieved molecular remissions in 8 of 11 of these patients. Data available at the time of this abstract for the 20-patient cohort, indicates a median follow-up of 9.167 years. 9 patients (45 %) remain in continuous first CR at their most recent follow-up (either in 2004 or 2005), and overall survival is 95%. The data further indicates the median disease free survival for the cohort is 96.5 months (8.04 years). To date there have been no additional reported mortalities in this cohort. As of August 2005, we report the progress of the Phase III clinical trial for this vaccine, opened in January 2000 by the NCI to evaluate the impact of this hybridoma-based Id vaccine on disease-free survival in a group of up to 375 previously untreated patients who have attained a CR or CRu from PACE [Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide (ProMACE without methotrexate)] chemotherapy, and who are randomized to receive either vaccine or control. To date, 187 patients have been accrued onto the study. Of those patients, 145 (77.5%) achieved a CR or Cru and are being followed in this ongoing clinical trial.

Strategies to enhance the specific anti-tumor effect of the graft without increasing the risk of graft-versus-host disease (GVHD) are needed to improve the outcome in allotransplant recipients. In this pilot study, we vaccinated sibling donors against a defined, patient tumor-derived antigen prior to bone marrow harvest. The objective of this strategy was to induce tumor antigen-specific immunity in healthy donors and to transfer this immunity to the recipients by bone marrow transplantation (BMT). For this purpose, the unique antigenic determinants (Idiotype [Id]) of the immunoglobulin secreted by the myeloma tumor can serve as a safe, tumor-specific antigen for active immunotherapy. The vaccine formulation consisted of Id isolated from the plasma of myeloma patients chemically conjugated to a carrier molecule keyhole limpet-hemocyanin (KLH) and administered together with GM-CSF as an adjuvant. Immunization of the HLA-matched sibling donors (n = 5) prior to bone marrow harvest, induced specific cellular and/or humoral immune responses against Id and KLH in all five donors. No short-term or long-term toxicities were observed in any of the donors following vaccination. The recipients received priming vaccination pretransplant and booster immunizations starting approximately 3 months after the myeloablative transplantation. Following BMT, two patients died within 30 days due to transplant related complications. Id-specific and KLH-specific T cell responses were detected in all three surviving patients post-, but not pre-BMT. In at least one patient, Id-specific T cell immunity persisted beyond 18 months after completion of the vaccination schedule. All three surviving patients converted from a partial response to a complete response following BMT. Two of the three patients (both recipients of immune BMT from syngeneic twins) remain in complete remission 8 years and 7 years after the transplant, and the third patient developed chronic steroid-dependent GVHD and died of renal failure after 5.5 years while in complete remission from the disease. Our results suggest that Id vaccination induces specific T cell immunity in the donors that is transferable by BMT, is associated with a prolonged disease-free survival in recipients and may represent a novel strategy to enhance the graft versus myeloma effect. These encouraging results have prompted us to initiate a clinical trial to evaluate this approach of donor immunization in the context of non-myeloablative allogeneic peripheral blood stem cell transplantation in myeloma patients.