A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

Background  

The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific).     

Human autologous tumor-specific T-cell responses induced by liposomal delivery of a lymphoma antigen.

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.     

A pilot study of CTLA-4 blockade after cancer vaccine failure in patients with advanced malignancy.

PURPOSE: Eleven patients with progressive advanced malignancy after administration of a cancer vaccine received a fully human anti-CTLA-4 monoclonal antibody (ipilimumab). The primary end point was to determine drug toxicity. Tumor response, tumor-specific CD8+ T-cell immune responses, and modulation of CD4+ CD25+ FoxP3+ regulatory T-cell (Treg) numbers were secondary end points. EXPERIMENTAL DESIGN: Three patients with colon cancer, four with non-Hodgkin's lymphoma, and four with prostate cancer were treated. The first dose was given at 3 mg/kg and subsequent doses were administered monthly at 1.5 mg/kg for a total of four cycles. RESULTS: Tumor regression was observed in two patients with lymphoma; one of which obtained a partial response of 14-month duration. Ipilimumab was well tolerated with predominantly grade 1/2 toxicities. One drug-related grade 3 toxicity was observed. One patient died within 30 days of treatment due to progressive colon cancer. No increase in vaccine-specific T-cell responses was observed after therapy. Tregs as detected by expression of CD4+CD25+CD62L+ declined at early time points but rebounded to levels at or above baseline values at the time of the next infusion. CONCLUSIONS: Ipilimumab treatment depressed Treg numbers at early time points in the treatment cycle but was not accompanied by an increase in vaccine-specific CD8+ T-cell responses in these patients previously treated with a variety of investigational anticancer vaccines. A partial response was observed in one patient with follicular lymphoma. A phase I/II trial evaluating ipilimumab in patients with follicular lymphoma is currently ongoing.     

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.     

Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles.

Idiotypic sequences, specific to the hypervariable regions of immunoglobulins expressed by malignant B cells offer a therapeutic target in B cell lymphoma. Efficient approaches have been described to clone a single chain fragment of the tumor immunoglobulin (Ig) comprising of heavy and light Ig chains (sFv) fused with proinflammatory chemokines. Tumor associated, poorly immunogenic self antigens encoded by plasmid DNA (pDNA) have been rendered immunogenic by chemokine fusion, thereby targeting to antigen presenting cells (APCs) which differentially express chemokine receptors. Here we present an injectable (parenteral) approach using synthetic polymer based cationic microparticle formulations for enhancing the potency of such chemokine/self antigen expressing plasmid construct. Branched and linear polyethyleneimine (PEI) were conjugated on poly (D, L lactide-co-glycolide) (PLGA) microparticles using carbodiimide chemistry followed by efficient loading of plasmid DNA. In addition to imparting significant buffering ability to these cationic microparticles, flow cytometry studies indicate that these DNA loaded microparticles significantly up regulate CD80 and MHC class II markers in phagocytic RAW264.7 cells, indicating intrinsic adjuvant effects. Intradermal injections in Balb/c mice with these formulations induced significant protection upon tumor challenge with 2.5 times the minimal lethal dose. Long term survival rates were significant (p < 0.05) in comparison with saline injected controls or blank microparticles. Further studies indicated that intramuscular delivery might provide better protection compared to intradermal injections and perform similar to gene gun mediated administration. We conclude, based on these promising in vivo results, that such surface-functionalized microparticles offer an attractive strategy to improve the potency of self antigen-based cancer DNA vaccines.     

A novel proteoliposomal vaccine induces antitumor immunity against follicular lymphoma

Clinical studies suggest that treatment with vaccines comprised of idiotype protein may be associated with improved clinical outcome in follicular lymphoma patients. The time-consuming process required to generate patient-specific vaccines is a major limitation, however. Here we report results of a pilot clinical trial with a novel autologous, tumor-derived proteoliposome vaccine formulation that could be rapidly produced within a single day. Vaccination was safe, induced autologous tumor-specific type 1 cytokine responses in 5 out of 10 follicular lymphoma patients, and was associated with induction of a sustained complete response in one patient. Other patients had large tumor burdens and progressed after a median duration of 8 months. These results suggest that further testing of this vaccine formulation, particularly in the setting of minimal disease, is warranted. Furthermore, the proteoliposome formulation may provide a model for vaccine development for other human cancers, for which tumor-associated antigens need not be defined.     

Survivin DNA vaccine generated specific antitumor effects in pancreatic carcinoma and lymphoma mouse models

We investigated the antitumor effect of survivin DNA vaccine in murine pancreatic and lymphoma models, and if xenogenic survivin can generate stronger immune response. We found that mice vaccinated with either human or mouse survivin DNA have significantly slower tumor growth and longer survival than those vaccinated with vector DNA. There was no significant difference between groups that received human and mouse survivin DNA. Lymphocyte infiltration was greater in tumors of mice immunized with survivin DNA than in tumors of control mice. We conclude that survivin DNA vaccine generated specific antitumor effects with increased lymphocyte infiltration at the tumor sites.     

Pentostatin,cyclophosphamide and rituximab for previously untreated advanced stage,low‐grade B‐cell lymphomas

We conducted a prospective phase II trial of pentostatin, cyclophosphamide and rituximab as initial therapy for patients with previously untreated advanced stage low‐grade or indolent B‐cell lymphomas (iNHLs). Of 83 evaluable patients, 91·6% attained an overall response and 86·8% a complete or unconfirmed complete response. The 3‐year progression‐free survival (PFS) and overall survival rates were 73% and 93%, respectively. The 3‐year PFS rate was significantly different for different diagnoses (P = 0·01): 83% [95% confidence interval (CI): 0·72, 0·96] for follicular lymphomas, 73% (95% CI: 0·54, 1·0) for marginal zone lymphomas and 61% (95% CI: 0·46, 0·81) for small lymphocytic lymphomas. The most common adverse events were haematological. Of 509 cycles of chemotherapy administered, grade 3 or 4 neutropenia was reported in 68 cycles (13% of cycles administered) and most frequently occurred during cycles 4–6. This is the first report demonstrating the effectiveness of pentostatin, cyclophosphamide and rituximab in patients with previously untreated iNHLs, including those over 60 years of age.