Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy

Purpose/results/discussion. Recurrent chromosomal translocations are common features of many human malignancies. While such translocations often serve as diagnostic markers, molecular analysis of these breakpoint regions and the characterization of the affected genes is leading to a greater understanding of the causal role such translocations play in malignant transformation. A common theme that is emerging from the study of tumor-associated translocations is the generation of chimeric genes that, when expressed, frequently retain many of the functional properties of the wild-type genes from which they originated. Sarcomas, in particular, harbor chimeric genes that are often derived from transcription factors, suggesting that the resulting chimeric transcription factors contribute to tumorigenesis. The tumor-specific expression of the fusion proteins make them likely candidates for tumor-associated antigens (TAA) and are thus of interest in the development of new therapies. The focus of this review will be on the translocation events associated with Ewing's sarcomas/PNETs (ES), alveolar rhabdomyosarcoma (ARMS), malignant melanoma of soft parts (MMSP) (clear cell sarcoma), desmoplastic small round cell tumor (DSRCT), synovial sarcoma (SS), and liposarcoma (LS), and the potential for targeting the resulting chimeric proteins in novel immunotherapies

Particle counts, size distribution, and morphology of stirring induced aggregates of a broad array of antibodies.

<p>10 mAbs (with known or predicted rates of clinical immunogenicity) were aggregated by stirring stress and then examined for their subvisible and visible aggregate content. A) Aggregates were quantitated by HIAC to determine the number and size range of particles present. Bar height represents the differential particle counts per ml in each size range (average of 3 runs). B) Aggregates images were captured on a Micro-flow Imaging System to evaluate the morphology of particles present. Representative images of the largest particles detected are shown. The size threshold indicates the lower size limit of the particles that were used for comparison. The aggregate content of the original mAbs (before stirring stress) is also shown and highlights that only a few small sized aggregates were detected.</p

The response of CD4⁺ T-cells in the IVCIA assay agrees with the rate of clinical immunogenicity for biotherapeutic mAbs.

<p>10 mAbs, with known rates of clinical immunogenicity, were evaluated in the IVCIA assay in a population of 50 healthy human donors over 5–8 days. mAb1 has not been tested in the clinic. Donors that responded by multiple readouts were evaluated for the most effective relative risk ranking. The percentage of donors that showed A) a positive T-cell proliferative response ([³H]-thymidine uptake) or B) an increase in the number (No.) of IL-2 secreting cells (Elispot) over the course of the entire study are displayed. Results were also combined to illustrate the percentage of donors that showed C) either a positive T-cell proliferation response or an increase in the concentration of IL-2 secreted (multiplex cytokine analysis) or D) a positive T-cell proliferation response and an increase in the number of IL-2 secreting cells are shown. Not all donors were tested for IL-2 for some samples (<i>grey circles</i>). A response was considered positive if the SI ≥ 2.0 (<i>p</i><0.05) for proliferation or number of IL-2 secreting cells or the SI ≥ 1.9 for IL-2 concentration (above the background response). mAbs are ordered approximately within each graph from the lowest to the highest response in the IVCIA assay. The scale bars at the top of each graph show the highest incidence of immunogenicity reported for each mAb in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159328#pone.0159328.t001" target="_blank">Table 1</a>. All rates are associated with diverse disease indications and assay testing platforms with variable sensitivity. <i>Black circles</i> represent duplicate experiments in different sets of 50 donors.</p

The IVCIA assay can be used to compare mAb lots from both the same manufacturer and from different manufacturers (biosimilars).

<p>A) Several different lots of Erbitux (2 lots), Remicade (2 lots) and Humira (5 lots) were tested in the IVCIA assay for a response at the early (20 h) phase (n = 12 donors), at 40 μg/mL. The concentration (pg/mL) of signature cytokines that were secreted 20 hours after stimulation is shown. B) Biosimilars, Humira and ABP 501, from two different manufacturers were compared in the IVCIA assay for the secretion of signature cytokines at the early (20 h) and late (7 day) phases (n = 4 donors), at 100 μg/mL. C) Multiple lots of another set of biosimilars, Herceptin and ABP 980, from two different manufacturers (which are both associated with a low rate of clinical immunogenicity), were assessed in the IVCIA assay at 40 μg/mL for T-cell proliferative responses on Day 7 only. No statistically different responses in the assay were observed between different lots of Herceptin and mAb5-B (p = 0.12). In all assays, the average response across the population tested was similar, although slightly different responses in specific donors could be observed. In all panels, <i>bars in shades of white and grey</i> show the average level of cytokine secretion at the early and late phases, and <i>purple bars</i> depict the average level of T-cell proliferation, across the population. <i>Colored and black circles</i> represent the response of individual donors and show the variability of the population tested.</p

Use of <i>In Vitro</i> Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics

<div><p>An <i>In Vitro</i> Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.</p></div