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Introduction

Immunomodulatory biologics (IMBs) are biotherapeutic entities that enhance or suppress the host immune response as a means of treating various cancers and complex immune disorders. With their targeted selectivity and superior clinical efficacy, IMBs represent an emerging therapeutic strategy and investment opportunity for disease intervention by many pharmaceutical and biotech companies. Novel biopharmaceutical IMBs entering into clinical development demand highly specialized cell-based assays to address biomarker requirements. As a result, flow cytometric laboratory testing is becoming increasingly important to the clinical evaluation of IMBs as it offers automated, high-throughput, multiparameter analysis of immune cell phenotype and effector function in complex biological fluids. Throughout this chapter we will focus on flow cytometric applications for immune monitoring in peripheral blood as a tool for biomarker analysis in the context of IMBs. Considerations for the validation and implementation of flow cytometric biomarker assays in global, multicenter clinical trials will also be emphasized.

Overview of Immunomodulatory Biologics

Immunotherapy via the administration of biologics targeting the modulation of immune responses has therapeutic utility in a broad range of clinical indications including diseases of immune dysfunction (i.e., Crohn’s disease, rheumatoid arthritis), chronic infection, and cancer [1]. In general, IMBs have an attractive pharmacological profile including high on-target effects, a long half-life, and broad extracellular fluid biodistribution which make them attractive biopharmaceutical candidates for use in disease intervention [2]. IMBs can target specific immune cell subsets, such as ipilimumab, which blocks CTLA4 (CD152) on T-cells, or target soluble immune mediators, such as infliximab, which binds the cytokine TNF to block its interaction with TNFR [1, 3]. As such, IMBs are commonly grouped into three main categories based on their modes of action and include: recombinant human cytokines (i.e., IL-2, GM-CSF, IFN-α, β, γ), antibody-based triggering IMBs (i.e., alemtuzumab, rituximab), and blocking IMBs (i.e., pembrolizumab). Cytokine mimicry, cell depletion, activation, and suppression of immune cell effector responses are but a few key examples of how clinically administered IMBs exert their therapeutic effect [1].

Modulation of immune cell subsets using agonistic or antagonistic monoclonal antibodies (mAbs) is perceived to be a major opportunity for the treatment of various solid tumor and hematological malignancies [3]. In particular, a developing pipeline of therapeutic IMBs has emerged targeting immune checkpoint receptors on T-cells. Building on the success of ipilimumab, next generation blocking IMBs targeting T-cell co-inhibitory receptor PD-1 (CD279) have recently received FDA approval and those targeting one of its ligands PD-L1 (B7-H1) are currently in clinical development. To date, anti-PD-1 mAb clinical trial results show promising efficacy and safety profiles in multiple tumor types with the hope of incorporation into various treatment regimens for long-lasting clinical benefit [4, 5]. As such, this has set the stage for other promising T-cell immunomodulatory approaches to cancer treatment including the development of mAb agonists to co-stimulatory molecules OX-40, GITR, and CD40L as a means to augment the proliferative capacity, activation, and effector cytokine function of CD4+ and CD8+ T-cells during anti-tumor responses [2].

Flow Cytometry as a Biomarker Assay Platform to Support the Clinical Development of Immunomodulatory Biologics

Because IMBs have a broad range of clinical applications, targets, and modes of action it is important to incorporate sensitive and robust biomarker assays tailored to the specific biologic and its intended cellular target(s) throughout the clinical development process. Target engagement, predictive, and pharmacodynamic (PD) biomarkers are all vital to monitoring the ability of IMBs to modulate the cells and/or signaling pathways involved in the initiation and termination of effective immune responses. Furthermore, these biomarkers in the context of clinical trial design offer valuable information on dose selection, on-treatment dynamics of immune therapy, and clinical risk monitoring for exaggerated pharmacology and adverse reactions.

Flow cytometry is an ideal biomarker assay platform for the clinical evaluation of IMBs (Table 1). It is a high-throughput and information rich analytical technology that uses fluorescent dye conjugated reagents to simultaneously resolve morphology, antigen expression, and effector function of individual cells present in complex biological matrices such as blood, bronchoalveolar lavage, and bone marrow aspirate. The technology works by hydrodynamically focusing thousands of fluorescent reagent(s) stained cells in suspension within a sample stream toward a laser-based interrogation point where beam steering optics direct emitted fluorescence from cells into photomultiplier tubes for conversion into digital signals by electronic components within the instrument. Digital signals are then converted into a standardized file format (.fcs) for visualization as histograms and/or bivariate dot plots using specialized software packages [6]. More advanced computational and visualization tools are also emerging for complex data sets. Most clinic facing cytometers to date are capable of measuring at minimum ≤10 biomarkers of relevant immune cell phenotype and/or effector function within a single patient sample. As such, flow cytometry is a highly versatile technology to support clinical biomarker analysis.

Table 1 Flow cytometric biomarker assays for the clinical development of immunomodulatory biologics
Full size table

Immunophenotyping

Extensive immune cell characterization and/or enumeration can be achieved in a single patient sample using a combination of direct/indirect staining methods with fluorochrome-conjugated mAbs against cell surface and intracellular antigens. Clinical laboratory testing of major T-, B-, and natural killer (TBNK) immune cell subsets can be accomplished using commercially available lyophilized mAb kits with reference ranges established for major peripheral blood leukocyte populations in healthy volunteers. As such, TBNK immunophenotyping is routinely employed as a safety and/or PD biomarker in clinical trials. For IMBs, TBNK analysis has been used to enumerate CD19+ B-cell counts pre- and post-treatment with rituximab, the anti-CD20 mAb, to determine treatment response and differentiate patient responders from non-responders [7, 8].

Immunophenotyping of major immune cell subsets can also be combined in parallel with other mAb to further differentiate cell subpopulations as treatment relevant biomarkers. A variety of phenotypic markers on T-cells including CD45 RA or RO, CCR7, CD25, CD127, FoxP3, HLA-DR are commonly multiplexed with major lineage markers to identify naïve, memory, regulatory, and activated T-cell subsets [9]. As an example, phenotypic analysis and enumeration of regulatory CD4+ T-cells (Tregs) differentially expressing CD25/CD127 and intracellular FoxP3 is becoming an increasingly important consideration in the development of IMBs as their presence can impact the effect of immunotherapy on disease pathology. For instance, Treg modulation is hypothesized to be a possible mechanism of action for anti-TNFα agents in various autoimmune diseases such as inflammatory bowel disease (IBD). To this effect, increased frequencies of Tregs in peripheral blood were detected only in IBD clinical responders treated with infliximab suggesting peripheral Treg levels may correlate with clinical response [10].

Functional Assays

Flow cytometric biomarker assays that characterize cell-mediated immune responses combine standard immunophenotyping with a functional measurement of cellular physiology such as surface protein expression, intracellular cytokine production, or cell signaling induced phosphorylation of transcription factors and kinases [6]. Functional biomarker assays have diverse applications including PD assessment toward modeling PK/PD relationships, proof of pharmacology, and/or mechanism of action in clinical trials.

Modulation of cell surface or intracellular proteins associated with cell activation or suppression can be used to monitor PD effects following immunotherapy. Percent expression or median fluorescence intensity measurements converted into molecules of soluble fluorescence (MESF) using reference beads for standardization purposes may be used to measure longitudinal changes in protein expression in patient-derived samples during the course of a clinical trial. In regard to IMBs, CTLA4 blockade with ipilimumab stimulates anti-tumor immune responses [3]. As such, increased expression of the activation marker, ICOS, and the proliferation marker, Ki67, on circulating CD4+ and CD8+ T-cells have been reported as PD biomarkers in melanoma patients following treatment with ipilimumab [11]. Other potential markers of T-cell activation may include HLA-DR, CD27, OX40, 4-1BB, and CD40L. In contrast, increases in BTLA, PD-1, LAG3, and TIM-3 markers may signify functional T-cell exhaustion and/or inhibition [3, 9].

De novo synthesis of intracellular effector cytokines or phosphorylation of cell signaling proteins can also be used as a measure of functional responsiveness to immunotherapy. This is accomplished by combining cell surface immunophenotyping with intracellular cytokine staining (ICS) or phosphoflow methods using fixatives and permeabilization agents to gain access to cell cytoplasmic and/or nuclear compartments prior to mAb staining. As such, ex vivo assessment of T-cell functional competence by ICS and phosphoflow analysis has the potential to identify treatment relevant biomarkers. For example, intracellular cytokine staining in melanoma patients following CTLA4 blockade with the mAb tremelimumab revealed increases in IL-17 cytokine producing CD4+ cells following ex vivo stimulation [12]. These TH17 cells were preferentially increased in patients that developed clinically relevant autoimmune toxicities after one round of immunotherapy, highlighting mechanistic and dose limitations to the clinically administered mAb. Similarly, phosphoflow analysis in tremelimumab treated melanoma patients showed alterations in CD4+ and CD8+ T-cell signaling pathways. Increases in pSTAT1, pSTAT3, pp38 along with decreases in pLck, pERK1/2, pSTAT5, demonstrated PD effects consistent with direct inhibition of T-cell signaling downstream of the T-cell receptor (TCR) complex; the suspected mechanism of action for CTLA4 blocking mAbs [13].

Receptor Occupancy

Flow cytometric assessment of cell surface receptors and measurement of receptor occupancy (RO) pre- and post-IMB administration provides valuable information to confirm target engagement, to inform on dosing intervals, and to develop PK/PD relationships in early clinical trials. Flow cytometric RO assays have been previously reported for the pre-clinical development of small molecule antagonists in peripheral blood but the application is also amenable to IMB clinical development [14]. RO can be monitored on any leukocyte subset in peripheral blood if the target receptor is normally expressed. Receptor dynamics should be considered as transient, low expressing, and/or internalizing cell surface receptors present challenges during RO assay development. RO may also be assessed in certain cases at the desired site of drug action. As example, RO on malignant myeloblasts in bone marrow aspirates collected from patients with refractory acute myeloid leukemia has been previously reported in phase I studies with anti-CD33 immunoconjugates [15].

Flow cytometric RO assays are esoteric in design and execution but commonly employ fluorochrome-conjugated mAbs that differentially recognize epitopes to semi-quantitatively measure unbound (free), IMB occupied and/or total target receptors pre- and post-drug administration. In some instances, antibody binding bead standards are additionally used to quantitate the antibody binding capacity (ABC) of the detection mAbs to their receptors as a correlate representation of the absolute number of target receptors expressed on the cell surface. When implemented in a clinical trial setting, data from flow cytometric RO assays is compared longitudinally, often reporting RO relative to pre-dose baseline levels following administration of the IMB.

As a clinical development example, two anti-CD86 mAb were employed to determine RO for belatacept in clinical trials, a second generation CTLA4 Ig (LEA29Y) which binds CD86 expressed on antigen presenting cells [16]. The primary anti-CD86 mAb chosen had a high binding capacity for its target receptor and was able to detect low levels of belatacept RO without displacing it which in general are desirable characteristics when choosing a detection mAb to assess target engagement. In contrast, a non-competitive anti-CD86 mAb clone was also identified; one that did not interfere with belatacept and as such was used to calculate the total number of target receptor molecules. Alternatively, saturating amounts of the biotherapeutic IMB under clinical evaluation may be employed to quantitate RO using indirect mAb staining methods. To this effect, saturation with an anti-PD-1 mAb (MDX-1106) under clinical evaluation in a Phase I study of patients with refractory solid tumors was used to quantitate occupied PD-1 receptor sites and the total number of available PD-1 binding sites [5]. Specifically, the ratio of change in mean fluorescence intensity (rather than percentage positive events) of CD3+ lymphocytes pre-incubated ex vivo with saturating amounts of an isotype control antibody (indicating in vivo binding) or anti-PD-1 (to detect total available binding sites) was used to estimate PD-1 RO [5].

Assay Validation of Flow Cytometric Biomarker Assays

Assay validation of flow cytometric biomarker assays is a complex process due to the limited post-collection stability of clinical specimens, a lack of quality control (QC) reference standards, and technical variations between analytical laboratories [17]. Because flow cytometric applications are varied, validation parameters will be somewhat different across assay types. As such, flow cytometric biomarkers are validated using the fit-for-purpose paradigm which offers flexibility in validation requirements to meet the intended use of the data generated in a resource-effective fashion [18]. At minimum, analytical validation using fresh specimens will include an assessment of post-collection specimen stability, intra-/inter-assay precision and intra-subject variability testing in order to differentiate the effects of post-collection specimen handling, sample processing (often involving manual procedures), and inherent biological variability from the IMB mediated pharmacological effects on clinical specimens. In some instances, commercial disease specimens may also need to be procured from biorepository vendors prior to analytical validation.

When fresh specimens are desired, post-collection stability is first established to determine biomarker stability limitations from the time of specimen collection to flow cytometric data acquisition. Once established, standardized instruction procedures for specimen collection, handling and shipping conditions are to be provided to all participating clinical study sites to ensure clinical specimen integrity within the assay stability window. Next, intra-assay (within-run) and inter-assay (between-run) precision is evaluated to measure variability in assay performance. For immune cell subsets that are at least 10 % of the parental cell population, intra- and inter- assay precision with a coefficient of variability (CV) less than 20 % is generally considered acceptable. In instances where rare cell populations (<10 %) such as Tregs are to be measured, a CV up to 30 % may be acceptable but acceptance criteria should always be defined in context of intended clinical data use [19]. To further limit variability, all participating laboratory analysts must be well-trained and demonstrate competency before participating in assay validation. Finally, an assessment of intra-subject variability (within donors) is conducted in order to identify potential diurnal variations. Once complete, final results from validation experiments must be documented and reviewed to ensure adequate confidence in the measurements.

Assay Implementation in Clinical Trials

In order to ensure consistent flow cytometric assay performance and high-quality biomarker data across all regional laboratories over the course of a global, multicenter clinical trial implementation of appropriate QC materials during clinical sample analysis and the effective management of analytical variables using a contract research organization (CRO) is vital to successful assay implementation.

QC materials are used to monitor sample processing and flow cytometric assay performance enabling analysts to capture analytical errors in real time, to resolve technical issues and perform reanalysis within specimen stability limits as needed. QC materials should mimic the clinical specimen type and express the biomarkers of interest in the expected target range. In some instances, commercially available preserved blood specimens with limited phenotype and stability over several weeks can be used to monitor clinical sample analysis. Alternatively, frozen peripheral blood mononuclear cells and/or cell lines which express biomarkers of interest may also be used as QC materials. Once a QC material is identified, the laboratory establishes a lot-specific QC acceptance range to qualify analytical runs by evaluating the 95 % confidence interval from 10 to 20 analyses. If lot-specific QC data falls out of the acceptance range during clinical sample analysis, biomarker assay results are to be reevaluated and, if necessary, the laboratory testing is to be repeated [17].

To meet the increasing demands for flow cytometric biomarkers in multicenter clinical trials, the placement of clinical sample analysis at CROs offers a significant degree of standardization. This is attributed to the CRO use of identical flow cytometry instruments, SOP-driven methods, and centralized peer review of data quality; all serving to minimize biomarker assay variation in the clinical data set [20]. When evaluating a CRO, a detailed review of scientific expertise and laboratory personnel balanced with an infrastructure assessment for supporting early and late stage global clinical trials is highly recommended. The scientific proficiency of a CRO should initially be assessed by means of a pilot study using a well-established biomarker assay developed by the sponsor group. Of note, routine communication between the sponsor and the CRO laboratory is vital to successful assay implementation [20].

Summary

Therapeutic IMBs represent an emerging strategy for disease intervention; in particular, for the treatment of various cancers using mAbs to stimulate host anti-tumor immune responses. As novel IMBs enter into clinical trials, flow cytometric immune monitoring is increasingly in demand to meet biomarker requirements for the clinical development of IMBs. Flow cytometric biomarker assays incorporating immunophenotyping, analyses of effector function and receptor occupancy are broadly utilized to confirm target engagement, explore PK/PD correlations, interogate mechanism of action, and predict treatment response to immunotherapy; all of which provide valuable information for guiding clinical decisions and supporting IMB filings with regulatory agencies. Like most cell-based assays validation and implementation of flow cytometric biomarker assays in global, multicenter clinical trials remains a complex process. A fit-for-purpose approach to assay validation ensures adequate confidence in biomarker measurements while assay implementation using CRO laboratories offers a significant degree of standardization for the effective management of analytical variables encountered during clinical sample analysis.

Summary Box

  • Immunomodulatory biologics (IMBs) represent an emerging strategy for disease intervention by pharmaceutical and biotech companies; in particular for the treatment of complex immune disorders, (i.e., Crohn’s disease, rheumatoid arthritis), chronic infection, and cancer.

  • Flow cytometric laboratory testing is becoming increasingly important to the clinical evaluation of IMBs as it offers automated, high-throughput, multiparameter analysis of immune cell phenotype, and effector function in complex biological fluids.

  • Flow cytometric biomarker assays are commonly used to confirm target engagement, explore PK/PD correlations, interrogate mechanism of action during clinical trials.

  • Flow cytometric biomarker assays are validated using the fit-for-purpose paradigm which offers flexibility in validation requirements to meet the intended use of the data generated in a resource-effective fashion.

  • Minimum requirements for the analytical validation of flow cytometric assays using freshly collected blood specimens include an assessment of post-collection specimen stability, intra-/inter-assay precision and intra-subject variability testing.

  • The identification and use of appropriate QC materials during clinical sample analysis and the effective management of analytical variables using a contract research organization (CRO) help ensure consistent flow cytometric assay performance and high quality biomarker data over the course of a global, multicenter clinical trial.