Jan L. Fisher

Phase Ia/Ib trial of bispecific antibody MDX-210 in patients with advanced breast or ovarian cancer that overexpresses the proto-oncogene HER-2/neu.

PURPOSE MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2.

A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

Immune response in patients with newly diagnosed glioblastoma multiforme treated with intranodal autologous tumor lysate-dendritic cell vaccination after radiation chemotherapy.

Patients with glioblastoma multiforme (GBM) are profoundly immunosuppressed and may benefit from restoration of an antitumor immune response in combination with conventional radiation therapy and temozolomide (TMZ). The optimal strategies to evaluate clinically relevant immune responses to treatment have yet to be determined. The primary objective of our study was to determine immunologic response to cervical intranodal vaccination with autologous tumor lysate-loaded dendritic cells (DCs) in patients with GBM after radiation therapy and TMZ. We used a novel hierarchical clustering analysis of immune parameters measured before and after vaccination. Secondary objectives were to assess treatment feasibility and to correlate immune response with progression-free survival (PFS) and overall survival. Ten eligible patients received vaccination. Tumor-specific cytotoxic T-cell response measured after vaccination was enhanced for the precursor frequency of CD4+ T and CD4+ interferon &ggr;-producing cells. Hierarchical clustering analysis of multiple functional outcomes discerned 2 groups of patients according to their immune response, and additionally showed that patients in the top quintile for at least one immune function parameter had improved survival. There were no serious adverse events related to DC vaccination. All patients were alive at 6 months after diagnosis and the 6-month PFS was 90%. The median PFS was 9.5 months and overall survival was 28 months. In patients with GBM, immune therapy with DC vaccination after radiation and TMZ resulted in tumor-specific immune responses that were associated with prolonged survival. Our data suggest that DC vaccination in combination with radiation and chemotherapy in patients with GBM is feasible, safe, and may induce tumor-specific immune responses.

Tracking antigen‐driven responses by flow cytometry: Monitoring proliferation by dye dilution

Cell‐tracking reagents such as the green‐fluorescent protein labeling dye CFSE and the red‐fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back‐calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen‐specific tetramer binding, cytokine production and T‐cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNγ in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen‐driven immunological responses.

Clinical and immunologic effects of intranodal autologous tumor lysate-dendritic cell vaccine with aldesleukin (interleukin 2) and IFN-α2a therapy in metastatic renal cell carcinoma patients

Purpose: To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-α 2a in metastatic renal cell carcinoma patients. Experimental Design: Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 × 107 cells/1 mL Lactated Ringers solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-α 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti–renal cell carcinoma antibody and cytokine levels. Results: All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4+ T regulatory cells had a strong association with outcome. Pre–IP-10 serum levels approached significance for predicting outcome. Conclusions: The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.

Immune modulation effects of concomitant temozolomide and radiation therapy on peripheral blood mononuclear cells in patients with glioblastoma multiforme

Concomitant radiation therapy (RT) and temozolomide (TMZ) therapy after surgery is the standard treatment for glioblastoma multiforme (GBM). Radiation and chemotherapy can affect the immune system with implications on subsequent immune therapy. Therefore, we examined the phenotype and function of peripheral blood mononuclear cells in 25 patients with GBM prior to and 4 weeks after treatment with RT-TMZ using multicolor flow cytometry, as well as in vitro CD4(+) regulatory T cell (T(reg)) suppressor and dendritic cell maturation assays. RT-TMZ induced significant lymphopenia, with a decrease in total CD4(+) T cells, but did not significantly change monocyte counts. The proportion of functional T(reg) cells increased after treatment, whereas their absolute numbers remained stable. There was also a measurable decrease in the proportion of CD8(+)CD56(+) and absolute number of CD3(-)CD56(+) effector cells. Posttherapy monocytes retained the ability to mature into dendritic cells. Treatment with RT-TMZ is associated with changes in regulatory and effector peripheral blood mononuclear cells that tilt the balance towards an immune suppressive state. This shift can affect the outcome of immune therapy following RT-TMZ treatment and should be considered in the design of future combination therapy regimens.

Multiparameter precursor analysis of T-cell responses to antigen

Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.

Ex vivo expansion of tumor specific lymphocytes with IL-15 and IL-21 for adoptive immunotherapy in melanoma

Although T central memory cells have been described as the most effective T-cell subtype against tumor growth, little is known about the requirements needed for their optimal ex vivo generation. Hence, our goal is to establish a protocol that will lead to consistent ex vivo generation of lymphocytes skewed toward a central memory phenotype. Antigen-specific T-cell lines were generated by ex vivo stimulation with Class-I and Class-II melanoma peptide pulsed dendritic cells in the presence of either IL-2 or IL-15 plus IL-21. Tumor specific lymphocytes of both central memory and effector characteristics were consistently generated from healthy donors and melanoma patients. IL15/IL21 cultures result in a cell population with a lower proportion of CD4(+)CD25(high)FoxP3(+) regulatory cells and higher number of CD8(+) and CD56(+) cells, and consequently render a higher yield of cells with a greater cytolytic activity and IFN-gamma production against melanoma cell lines.

Humanized mAb H22 binds the human high affinity Fc receptor for IgG (FcgammaRI), blocks phagocytosis, and modulates receptor expression.

About 10–15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high‐dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcγR), because several model systems clearly show that functional FcγR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcγR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcγRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcγRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcγRI through its Fc domain and block FcγRI‐mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross‐linking FcγRI with mAb H22 rapidly down‐modulated FcγRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcγRI ligand binding domain and down‐modulates FcγRI expression, it is a particularly good candidate for evaluating the role of FcγRI in patients with ITP. J. Leukoc. Biol. 62:469–479; 1997.

Advantages of hydrophobic culture bags over flasks for the generation of monocyte-derived dendritic cells for clinical applications

Dendritic cells (DC), potent antigen presenting cells capable of activating both naïve and primed T cells, are currently being pursued clinically in the development of cancer vaccines. Variations in the literature regarding DC source, culture conditions, maturation state, dose, and route of immunization make comparisons of clinical trial data difficult. In order to define and optimize the culture conditions for DC generation, we have performed a careful comparison of two culture methods, as well as different methods of DC maturation. Our studies demonstrate that high viability DC can be produced and matured in gas permeable hydrophobic culture bags. These cells express surface molecules characteristic of DC and have superior yield, viability, and function to cells cultured in plastic tissue culture flasks. These results suggest that hydrophobic culture bags are ideal for the preparation of clinical DC vaccines, as DC can be generated, antigen-loaded, and matured in a closed system, a scheme we have found to be superior to previously described flask culture methods.