"Monitoring" refers to serial specimen acquisition and testing. The rationale for immune monitoring rests on the premise that therapeutic interventions achieve their effects as a result of modification(s) in one or more components of the patient's immune system. These therapy-induced modifications occur gradually, and the expectation is that by serially measuring immune biomar-kers that undergo changes relative to the pretherapy baseline level, it might be possible to define immunologic mechanisms responsible for biologic and possibly also clinical activity of the therapeutic agent. As biologic agents have a bell-shaped activity curve that shifts depending on the dose and time of their delivery, serial monitoring is necessary to define the optimal biologic dose (OBD) of a therapeutic agent, which often is distinct from the maximal tolerated dose (MTD). The latter is utilized to define toxicity of drugs, but because most biologic agents have no or little toxicity, the OBD is the appropriate measure of their effects. Since, however, biologic agents are likely to have multiple biologic (and clinical) effects, the definition of OBD may not be straightforward, depending on more than one immunologic assay. The major objective of serial immune monitoring is to establish a correlation between phenotypic and/or functional changes in immune cells induced by therapy and clinical responses. The major unanswered question, however, concerns the origin of immune cells to be tested. Peripheral blood mononuclear cells (PBMC) representing less than 2% total body mono-nuclear cells are most commonly employed, although it appears that cells derived from the disease site (e.g., site of infection, tumor, tumor-draining lymph nodes, fluid from an inflamed joint, or interstitial fluid from injured sites) best reflect the extent of alterations induced by disease. Thus, whenever available, such specimens should be collected, banked, and evaluated in parallel with peripheral blood.
A brief description of requirements that underlie the principles of immune monitoring is provided to orient the reader. specimens collected from subjects prior to, at defined intervals during, and at the end of therapy are delivered to the laboratory. The specimens usually consist ofperipheral blood collected into heparinized tubes, but may include tumor or other tissues, body fluids (e.g., pleural or peritoneal fluids, ascites), as well as especially collected interstitial fluids from sites of cannulation . The specimens for immune monitoring are harvested at intervals specified in the clinical protocol and have to arrive at the laboratory no later than 24 hours after harvest. This requires an overnight delivery of specimens originating at a distant location. The specimens are bar-coded and processed immediately upon arrival. The separated cells are either cryopreserved at —80 °C in 2 ml cryovials and banked or are immediately tested in assays that cannot be performed with cryopreserved/thawed cells. The monitoring laboratory is cognizant of assays that have to be performed on fresh as opposed to cryopreserved/thawed cells and will be prepared to handle the specimens accordingly.
Changes occurring in the immune cell phenotype or function in response to therapy may be difficult to detect, unless sensitive and reliable monitoring assays are available. To decrease interassay variability of assays, immune monitoring is generally performed with "batched" specimens, representing the entire collection of samples obtained from one subject throughout therapy. This type of design mandates that all collected specimens be cryo-preserved under controlled conditions, thawed with a minimal loss of viability, and tested in the same assay. It also requires that the monitoring laboratory has the capability to perform cryopreservation, bank, and maintain samples at a large scale for prolonged periods of time. An assay "reliability" in this context depends on the selection for monitoring of those immune markers/functions that are least affected by cryopreservation/thawing. This has to be a priori ascertained by the monitoring laboratory through comparisons of fresh and frozen specimens tested in the same assay. Experience shows that the correctly performed process of freezing/thawing of immune or other cells is by far the most crucial determinant for preserving their true functional potential and, hence, for successful monitoring.
Immune monitoring of clinical trials is a complex and demanding enterprise requiring considerable resources. Its translational role in bridging basic immunologic insights with clinical endpoints, however, cannot be overemphasized. There is increasing awareness of the fact that biologic therapies occupy an important place among available clinical modalities for treatment of human disease. Their true impact on disease processes cannot be unraveled without a better understanding of immune mechanisms that these therapies target and possibly alter. Without reliable immune monitoring to help identify and define these mechanisms, biotherapy is unlikely to achieve a strong scientific foundation it deserves. Additionally, immune biomarkers identified by well-done monitoring might well prove to be significant surrogates of disease development, activity, or progression, and as such play a key role in clinical practice. Immune monitoring represents a valuable component of future research in translational science and clinical medicine.
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