Regulatory perspective on in vitro potency assays for human dendritic cells used in anti-tumor immunotherapy

ARTICLE IN PRESS Cytotherapy, 2018; 0001 20

Regulatory perspective on in vitro potency assays for human dendritic cells used in anti-tumor immunotherapy

CHARLOTTE DE WOLF1,2, MARJA VAN DE BOVENKAMP1 & MARCEL HOEFNAGEL1 1

Medicines Evaluation Board College ter Beoordeling van Geneesmiddelen-Medicines Evaluation Board (CBG-MEB), Utrecht, The Netherlands, and 2Department of Infectious Diseases and Immunology, Utrecht University, The Netherlands Abstract Dendritic cells (DCs) are key connectors between the innate and adaptive immune system and have an important role in modulating other immune cells. Therefore, their therapeutic application to steer immune responses is considered in various disorders, including cancer. Due to differences in the cell source and manufacturing process, each DC medicinal product is unique. Consequently, release tests to ensure consistent quality need to be product-specific. Although general guidance concerning quality control testing of cell-based therapies is available, cell type-specific regulation is still limited. Especially guidance related to potency testing is needed, because developing an in vitro assay measuring cell properties relevant for in vivo functionality is challenging. In this review, we provide DC-specific guidance for development of in vitro potency assays for characterisation and release. We present a broad overview of in vitro potency assays suggested for DC products to determine their anti-tumor functionality. Several advantages and limitations of these assays are discussed. Also, we provide some points to consider for selection and design of a potency test. The ideal functionality assay for anti-tumor products evaluates the capacity of DCs to stimulate antigen-specific T cells. Because this approach may not be feasible for release, use of surrogate potency markers could be considered, provided that these markers are sufficiently linked to the in vivo DC biological activity and clinical response. Further elucidation of the involvement of specific DC subsets in anti-tumor responses will result in improved manufacturing processes for DC-based products and should be considered during potency assay development.

Key Words: dendritic cells, government regulation, immunotherapy, in vitro potency assays, neoplasms, quality control

Introduction Many anti-tumor approaches are currently based on targeting specific (immune) cells or pathways that play a significant role in reducing or enhancing tumor burden. These targeted immunotherapies include monoclonal antibodies, tyrosine kinase inhibitors and so-called ‘therapeutic vaccines’ [1,2]. The latter can use irradiated patient-derived or allogeneic tumor cells or (genetic material encoding) tumor-specific antigens [3 5]. To induce an antitumor immune response against these antigens, they can be administered in a viral vector or in combination with immunostimulating factors. But the use of (autologous) ex vivo cultured and/or modified immune cells may be the most promising and effective therapeutic anti-tumor approach. Nowadays, several immune cell types are (pre-)clinically tested or even applied for a large variety of tumors. These include, but are not limited to antigen-specific T effector cells, allo-reactive natural killer (NK) cells

and antigen-presenting dendritic cells (DCs) (e.g., [6 8]). The latter present antigen after ex vivo exposure to tumor lysate, loading with tumor proteins or peptides, transduction with genetic material encoding these tumor-associated antigens or fusion with inactivated tumor cells [7,9,10]. Recently, we have reviewed in vitro potency (i.e., functionality) assays for characterisation and release testing of cell-based therapeutic products. Thereby, cellular capabilities required for clinical application of immunomodulating mesenchymal stromal cells and anti-tumor T cells have been discussed [11,12]. In this review, we will concentrate on DCs as antitumor treatment and the in vitro control of their functionality during manufacture.

DC function and subsets DCs are professional antigen-presenting cells (APCs), which can generate a robust immune

Correspondence: Marcel H.N. Hoefnagel, PhD, Postbus 8275, 3530 RG Utrecht, The Netherlands. E-mail: [email protected] (Received 13 March 2018; accepted 14 July 2018) ISSN 1465-3249 Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2018.07.006

ARTICLE IN PRESS 2 C. DE Wolf et al. response and in the meantime maintain tolerance to harmless antigens [13]. Because DCs are considered to play a key role in the connection between the innate and adaptive immune system and are also important in the modulation of other (innate) immune cells, they are used in numerous immunotherapeutic approaches in the attempt to control the -dysregulated- immune response [7,9]. This control involves induction and modulation of other immune cells to, for example, recognise and attack tumor cells or diminish auto-reactivity. Several different DC subsets from the myeloid and lymphoid lineage are present in various tissues in the body (e.g., [14,15]). Each DC subtype harbors its own repertoire of overlapping and unique surface markers, functional specialisation (e.g., cross-presentation) and cytokine/chemokine production [16]. The efficacy of DC-based immune therapies mainly relies on activation of a proper T cell response, induced via the major histocompatibility complex (MHC)-peptide complex, co-stimulatory molecules and secreted cytokines. DC-derived cytokines are essential for activation of cytotoxic T cells (CTL, CD8+) and polarisation of na€ıve T cells into the correct T helper (Th, CD4+) cell type. For example, interleukin (IL)-12 induces differentiation towards a Th1 response, whereas DCs not producing IL-12 usually stimulate T cells to become Th2 cells [13]. Felzmann et al. showed that IL-12 producing DCs could efficiently trigger cytolytic activity in autologous T cells and that IL-12 was indispensable for CTL activity [17]. In addition, Felzmann et al. and Luger et al. found that IL-12 production was limited to the first 24 h after exposure to a maturation stimulus [17,18]. DCs subsequently converted into an anti-inflammatory phenotype. This reflects the in vivo situation where DCs and T cells first have contact in the lymph node and then specifically activated T cells migrate to the site of inflammation before DCs convert to an anti-inflammatory phenotype. DCs can also have direct cytolytic activity via the production of cytotoxic effector molecules, either in their immunoregulatory role (i.e., lymphocyte killing activity) or to remove malignant cells (i.e., tumoricidal activity) [19]. The combination of this killing capacity and antigen presentation has been described for natural as well as ex vivo induced DCs, and makes these so-called killer DCs even more interesting tools for immunotherapy [19].

DC product development Clinical application DC-based treatments have their origin in the fight against malignancies. In 2017, more than 700

clinical studies using DCs as immunotherapy were registered, of which approximately 75% had an oncological indication (www.clinicaltrials.gov). The remaining 25% of the studies focussed on effectiveness of DCs in the treatment of auto-immunity (mainly diabetes mellitus, rheumatoid arthritis and multiple sclerosis) and infections (primarily human immunodeficiency virus [HIV]). So far, DC-based anti-tumor treatments have been tested for a variety of malignancies, such as melanoma, prostate cancer, renal cell carcinoma, breast tumors and malignant glioma. Despite the potent antigen-presenting and T cell stimulating capacity of DCs, the clinical response in a significant proportion of patients has been disappointing [20]. Therefore, numerous approaches are in development to improve the stimulatory capacity of DCs or to combine DC treatment with other strategies to enhance efficacy, as discussed below [20,21]. Such improvements in DC therapy should also be considered in the setup of a potency assay.

Manufacture of DC products for anti-tumor therapies DC source Most DC-based immunotherapeutic approaches make use of (relatively easily accessible) bloodderived monocytes to ex vivo differentiate into monocyte-derived DCs (moDCs; Figure 1). Various protocols for the generation of moDCs exist. Most make use of culture with granulocyte macrophage colonystimulating factor (GM-CSF) and IL-4 [7,9,22]. However, also blood-derived natural DCs or CD34+ precursor cells cultured in the presence of, for example, GM-CSF or peripheral blood mononuclear cells (PBMCs) transmigrated through endothelial cells are proposed approaches for ex vivo DC generation [7,9,23] (Figure 1). In human blood, two major natural DC types are present: plasmacytoid DCs (pDCs) and myeloid DCs (myDCs) [9,24]. These DC types differ in expression of surface markers, migration patterns and functions. The DC subsets act synergistically for optimal immune responses, for example, the cells can activate each other and augment the expression of co-stimulatory molecules [25]. Natural bloodderived DCs were found to be superior APCs compared with moDCs [26]. Cells applied as immunotherapy can be either autologous or allogeneic (Figure 1). The use of allogeneic cells is one of the approaches to potentially promote immunogenicity toward tumor antigens. Allogeneic DCs are known to induce CD4+ T cells, which subsequently can promote bystander specific T cell induction [27]. Allogeneic pDCs (primary

ARTICLE IN PRESS Regulatory perspective on potency assays for anti-tumour dendritic cells

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Figure 1. DC manufacture, characterisation and release. Manufacture: pro-inflammatory DCs can be cultured from allogeneic or autologous blood-derived cells. DCs are subsequently genetically transduced (optional step), matured and loaded with antigen. Then, cells are washed and formulated (and stored) before injection into the patient. Throughout the manufacturing process, several in-process controls (IPCs) are implemented to warrant consistent production. Characterisation: DC functionalities other than those measured during batch release should be covered in characterisation studies. These include migration (toward chemokines) and maturation (e.g., surface marker and cytokine expression). Also selection and design of the most relevant potency assay, (surrogate) read-outs for release and the corresponding acceptance criteria is based on characterisation (and non-clinical) data. Release: consistency of several product characteristics should be shown during release testing. For DC potency, analysis of (surrogate markers for) antigen-specific T cell induction is expected. This setup will allow simultaneous analysis of antigen presentation, co-stimulation and polarisation (these DC functions could also be separately analysed during characterisation). The tumor-attacking capacity of stimulated T cells requires attention in characterisation studies. DC key functionalities that should be investigated in process validation, characterisation or release are depicted in red. Assays for characterisation may also be implemented for release, when required.

cells or derived from a cell line) loaded with tumor antigens are able to elicit a strong antigen-specific CD8+ T cell response, as shown both in vivo (humanised mice) and in vitro (cells from patients and healthy donors) [27,28]. These CD8+ T cells produced cytokines and induced lysis of tumor cells (in vitro) or reduction of tumor size (in vivo) [27,28]. Moreover, the used allogeneic pDC cell line was found more effective in activating functional antigenspecific CD8+ T cells than autologous or allogeneic myDCs [27]. The pDC cell line could be even

further activated by irradiation of these cells, which resulted in augmentation of co-stimulatory molecule expression on the cell surface [27]. DC maturation and polarisation Maturation of DCs can occur via addition of specific antigens and/or (a mix of) factors such as tumor necrosis factor (TNF)-a, prostaglandin E2, interferon (IFN)-a or -g, IL-1b, IL-6, different Toll-like receptor agonists or CD40L(-expressing cell lines) [3,5,9,22,29 32] (Figure 1). Mature DCs have an

ARTICLE IN PRESS 4 C. DE Wolf et al. activated phenotype, respond to lymph node homing signals and secrete Th1-polarising cytokines (e.g., IL-12). Most DC-based therapies developed so far primarily focus on activation of antigen-specific CD8+ T cells. However, for an efficacious anti-tumor therapy, the induction of both CD8+ and CD4+ T cell responses with the elimination of suppressor cell responses (e.g., CD4+ regulatory T cells [Tregs]) and the breakdown of the immunosuppressive tumor micro-environment is required [3,5,30,33]. Thus, DCs that are specifically designed to generate a multi-cell type immune response may increase the immunotherapeutic efficacy. Teramoto et al. proposed to load DCs with both MHC class I and MHC class II tumor peptides, resulting in the activation of IFN-g producing CD4+ T cells (with reduced numbers of CD4+ Tregs) and augmented CTL responses [34]. Other DC manipulations To positively influence the micro-environment at the site of injection, DC products can also be combined with adjuvants [3,5,7,30,35]. In some cases, DCs are transduced with a genetic construct to express specific cytokines or surface markers to further increase their T cell activating potential and even overcome immune suppressive mechanisms (Figure 1), although this is usually attempted by combining DCs with other types of immunotherapy (e.g., [3,5,21,36,37]). DC genetic transduction can also be applied to stably express tumor antigens [7,38,39]. This is expected to lead to DCs with efficient peptide presentation on MHC class I molecules, leading to potent CTL induction. Especially the addition of intracellular antigen-targeting approaches or DC survivalpromoting mediators is thought to induce better CTL responses with higher avidity for tumor antigens [39,40]. As said, DCs are usually ex vivo-cultured or expanded, exposed to tumor cell lysate or loaded with tumor-associated antigens and then re-introduced in the patient [9,10,21,22,38,41]. Next to this ex vivo culturing and antigen loading of DCs, the in vivo DC activation is an emerging approach that may be a less time-consuming alternative (i.e., off-theshelf product) [3,7,9,10,29,30,33]. Some suggest that the use of only DC-derived exosomes (as an acellular immunotherapy) may be sufficient to induce an effective anti-tumor response [7,10,42]. But because the in vivo activation and exosome approaches are not yet frequently presented in the literature, we will only focus in this review on ex vivogenerated and/or expanded antigen-presenting DC products.

Control of DC manufacture A variety of approaches in the manufacture of DC products is available. The lack of standardisation in DC culture, antigen loading and maturation procedures used and the variability in (autologous) starting material make it challenging to define a standardised approach to ensure consistent product quality. Furthermore, the method of DC generation and stimulation may not only impact the desired immune cells, but may also lead to activation of suppressor cells in the DC product [3,37], which could, for example, affect the susceptibility of tumors for lysis by CTLs. Therefore, special attention should be given to the presence of cellular impurities and the consequence of their presence (or their in vivo induction) for DC functionality. Both the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) have provided guidance for manufacture and control of cell-based therapies [43,44]. Nevertheless, cell typespecific regulation for quality control testing is still limited. Especially guidance related to the assessment of potency of the cells is needed, because defining the cell type-specific properties that are relevant for its in vivo functions and the development of appropriate assay(s) to characterise and control these properties is challenging [44,45]. From our regulatory experience, we recognise that companies take potency assay development serious. However, assay development may benefit from more in-depth product characterisation. To provide further guidance related to potency, we recently have discussed the main requirements for in vitro potency assays and prerequisites for the functionality of cell-based therapeutics (with the focus on T cell based products) [12]. In this review, we provide a broad overview of in vitro potency assays suggested for characterisation and release of different DC-based medicinal products to determine their anti-tumor function. The tests evaluated in this review were selected based on assay types and read-outs proposed for autologous and allogeneic DC products in development during scientific advice, complemented with assays discussed in the literature that we considered relevant in the context of DC manufacturing control. We discuss several advantages and limitations of these in vitro assays (summarised in Table I). Although several assays have been performed with murine primary cells or cell lines, the assay principles and techniques may also be used for in vitro tests with human cells.

In vitro assays to test for DC functionality DCs produced for anti-tumor immunotherapy should have a stable pro-inflammatory signature and

Table I. Overview of the advantages and limitations of frequently proposed in vitro potency assays. Responder cells

Additional Read-out parameter stimuli or cells

DC antigen uptake assay

—

(Im)mature moDCs

Fluorescently labelled beads or particles

DC maturation assay

—

PBMCs

Tumor antigen fused to GM-CSF

—

&

moDCs Blood-derived DCs &DCs derived from different source (e. g., murine bone marrow)

&

moDCs

&

&

CD40L-expressing cell line (murine or human)

Single or dual maturation factors (e.g., TNF-a, LPS and/or IFN-g) & Cytokine cocktail &Nothing added (next to antigens)

Extent of bead/particle uptake (reduction upon maturation)

Advantages

Limitations

Key references

&

&

[23, 31, 41, 47]

Very short assay (few hours). Assay measures a marker important to determine the APC activation status. &Analysis at single cell level. &Simultaneous phenotypic characterisation of responder cells possible. &Short assay (around 2 d). Expression of cell surface &Assay measures important markers proteins (e.g., CD54, CD86, MHC class II) involved in APC activation status (e.g., co-stimulatory capacity). &Can easily be combined with determination of secreted soluble mediators in supernatant. &Analysis at single cell level. &Simultaneous phenotypic characterisation of responder cells possible (e.g., which APC is CD54+). & & Expression of cell surface Short assay (1 2 d). &Assay measures important markers proteins (e.g., CD80, CD83, CD86, CCR7, involved in APC activation status DC-SIGN, MHC class II, (e.g., polarisation, co-stimulatory PD-L1) and chemotactic capacity). &Intracellular cytokine gene &Analysis at single cell level (flow expression (mRNA of, for cytometry). & example, CCL17, IL-10, Simultaneous phenotypic characIL-12p35, IL-12p40) terisation of responder cells possi&Secretion of soluble mediable (flow cytometry). tors (e.g., IL-1b, IL-6, IL10, IL-12p40, IL-12p70, IP-10) in supernatant

Single matu- Secretion of soluble mediaration factor tors (especially IL-12p40 (e.g., IFNor IL-12p70) in g) supernatant &Cytokine cocktail

&

Assay measures only surrogate marker of functionality. &Assay measures only one (immature) DC effector function. &In vivo relevance of bead/particle as antigen is not clear.

&

Assay measures only surrogate marker [48] of functionality. &Read-out may not be directly related to T cell functionality (in vivo).

&

Assay may not be based on antigen [16 18, specificity (i.e., no specific antigen 31, 35, involved). 47, 55, &Assay measures only surrogate markers 74]a of functionality. &Read-outs may not be directly related to T cell proliferation (in vitro). & Intracellular transcription is not necessarily similar to protein production and secretion. &Analysis at population level (PCR and ELISA). &No simultaneous phenotypic characterisation of responder cells possible (PCR and ELISA). &Short assay (1 2 d). &Assay may not be based on antigen [32, 51] &Assay measures important markers specificity (i.e., no specific antigen involved). involved in APC activation status &Assay measures only surrogate marker (e.g., polarisation). &Assay can discriminate between difof functionality. ferent DC types that produce vari- &Assay can lead to misleading results ous levels of IL-12. when other secreted cytokines or sur&Read-out concentration correlates face markers affect IL-12 production or its function (on T cells). with activation of IFN&Read-out concentration may not g producing T cells (in vitro) and clinical outcome. correlate with frequency of antigenspecific T cells (in vivo). (continued on next page)

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Stimulator cells

Regulatory perspective on potency assays for anti-tumour dendritic cells

Proposed in vitro potency assay

5

Proposed in vitro potency assay

Stimulator cells

Responder cells

Additional Read-out parameter stimuli or cells

Advantages

Limitations

Key references

&

DC migration assay (transwell system)

&

Chemokines moDCs (CCL19, bmDCs &Tissue-derived DCs CCL21)

Extent of transmigrated DCs

&

—

PBMCs (containing immature monocytes)

&

—

moDCs

Detection of cell surface TCRmimics MHC class I molecules and maturacontaining intracellular tion processed peptide stimulus

Human endothelial monolayer on a matrix &Optional: antigen, adjuvant, etc.

Secretion of soluble mediators (e.g., IL-6, TNF-a) in supernatant

(continued on next page)

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Antigen presentation assay

—

In vivo relevance of murine cell line as stimulator is not clear. &Analysis at population level. & No simultaneous phenotypic characterisation of responder cells possible. &Very short assay (few hours) &Assay is not based on antigen specific[35, 61, &Assay measures a marker ity. 62]a &Assay measures only surrogate marker important for T cell induction (lymph node homing potential). of functionality. &Analysis at single cell level. &Assay measures only one DC effector & function. Simultaneous phenotypic &Autocrine chemokine production may characterisation of responder cells possible. interfere with migration. &Assay measures a marker important &Relatively long assay (around 3 d). [63]b &Assay may not be based on antigen for T cell induction (lymph node homing potential). specificity. & & Assay simulates relevant in vivo Assay measures only surrogate marker immune activation steps and transof functionality. &Assay measures only one DC effector migration across endothelium. &Assay can simulate in vivo changes function. &Assay requires the use of different cell in the micro-environment. &May be combined with additional types. &Influence of (migrating) cells other characterisation of cells (when properly isolated after migration). than DCs (monocytes) is not taken into account. &Assay can lead to misleading results when endothelial cells also secrete soluble mediators. &Analysis at population level. & No simultaneous phenotypic characterisation of responder cells possible. &Assay measures a marker &Relatively long assay (around 4 d). [65] &Assay measures only surrogate marker important for T cell induction. &Assay is based on antigen of functionality. &Assay measures only one DC effector specificity. &Density of MHC-peptide comfunction. &Assay does not take the effect of DC plexes on DCs correlates with in vivo CTL response. co-stimulation and cytokine secretion &Indirect correlation between on T cell activation into account. &Assay requires donor-specific (i.e., MHC-peptide complexes on DCs and in vitro antigen-specific T cell HLA type- and peptide-specific) response (i.e., reduction in case of TCRmimics. TCRmimics as MHC blockade). &Analysis at single cell level. &Simultaneous phenotypic characterization of responder cells possible.

6 C. DE Wolf et al.

Table I (Continued)

Table I (Continued) Responder cells

Additional Read-out parameter stimuli or cells

Co-stimulation assay

moDCs

Allogeneic T cells (standard batch)

Sub-optimal concentration of antiCD3

T cell priming or stimulation assay (autologous)

&

Irradiated moDCs (Weekly refreshed) moDCs &DCs derived from different source (e. g., murine bone marrow)

&

PBMCs (Na€ıve) T cells &Tumor-associated Tregs

&

&

&

Cord blood derived DCs

T cells

IL-2 and PHA T cell proliferation via MTT

IL-2, IL-7 and IL-15 (alone or in combination) &Nothing added

Advantages

Limitations

T cell proliferation via 3[H] Thymidine incorporation

&

&

&

&

Amount of antigen-specific (cytokine-secreting) T cells &Expression of T cell subset markers (e.g., CD4, CD8, CD56) &Expression of T cell surface proteins (e.g., CD69, CD154) &Intracellular cytokine production (e.g., IFN-g, IL-2) &Secretion of cytokines (e.g., IFN-g, IL-12p40, IL12p70, IL-17A) in supernatant &T cell proliferation via MTT, BrdU or 3[H]Thymidine incorporation &(Inhibition of) Treg suppressive capacity

Short assay (around 2 d). Assay measures a marker important for (na€ıve) T cell induction. &Lower assay variability compared with standard MLR. &May be combined with determination of secreted soluble mediators in supernatant. &

Assay makes use of autologous cells. &Assay is based on antigen specificity. &Assay measures functional cross-talk between DCs and responder cells. &Analysis at single cell level (flow cytometry). &Simultaneous phenotypic characterization of stimulator and responder cells possible (flow cytometry).

&

Assay makes use of autologous cells. &Assay measures functional crosstalk between DCs and responder cells.

Key references

(continued on next page)

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[66, 67] Assay measures only one DC effector function. &Read-out is not directly correlated with presented antigens. & Read-out is only semi-quantitative (i.e., physiologically relevant level of potency is unknown). &Analysis at population level. &No simultaneous phenotypic characterisation of stimulator and responder cells possible. &Very long assay (10 d to several weeks), [17, 23, except for most murine assays (around 35, 36, 4 d). 41, 47, &Assay can lead to misleading results 68, 74]a during long culture period (due to cell death or functional alteration of cultured cells). &Intracellular protein production is not necessarily similar to secretion. &Assay requires high amount of stimulator cells (in case of >1 stimulation round). & Effect of cytokine addition on DC functionality is not clear. &Effect of irradiation on DC functionality is not clear. &High assay (i.e., inter-donor) variability possible. &Analysis at population level (except for flow cytometry). &No simultaneous phenotypic characterisation of stimulator and responder cells possible (except for flow cytometry). &Cell source of secreted cytokines can not be determined. &Relatively long assay (3 d). [38] &Assay is not based on antigen specificity (due to PHA). &Effect of (significant amount of) IL-2 addition on DC and T cell functionality is not clear. & High assay (i.e., inter-donor) variability possible. &Analysis at population level. &No simultaneous phenotypic characterisation of stimulator and responder cells possible.

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Stimulator cells

Regulatory perspective on potency assays for anti-tumour dendritic cells

Proposed in vitro potency assay

Proposed in vitro potency assay

Stimulator cells

Responder cells

Additional Read-out parameter stimuli or cells

Advantages

Limitations

T cell stimulation assay (HLAmatched)

&

&

&

&

&

moDCs Blood-derived DCs &DC-derived exosomes & DC cell line &Irradiated tumor cell line-derived DCs &PBMC-derived APCs (after culture with tumor antigen &

(Patient- or healthy donor derived) PBMCs &TILs & Antigen-specific primary T cells &Antigen-specific T cell line &T cell hybridoma

fused to GM-CSF) or specific subsets &

&

&

&

moDCs MitomycinC treated moDCs & Skin-derived DCs &Irradiated tumor cell line derived DCs &Irradiated PBMCderived APCs (after culture with tumor antigen fused to GM-CSF) or specific subsets

Allogeneic PBMCs Allogeneic T cells (from one or more donors)

&

Amount of antigen-specific T cells &Expression of T cell subset markers (e.g., CD45RA, CD62L) &Intracellular cytokine production (e.g., IFN-g) &Secretion of cytokines (e.g., IFN-g, IL-2) in supernatant

Short assay (around 1 2 d), except for [27, 28] (>7 d). &Assay is based on antigen specificity (with dose-dependent response). &Assay measures functional crosstalk between DCs and responder cells. &Analysis at single cell level (flow cytometry). & Simultaneous phenotypic characterization of stimulator and responder cells possible (flow cytometry). &Cytokines (e. &Expression of intracellular &Assay measures functional g., IL-2) factors (e.g., FoxP3) cross-talk between DCs and &Nothing &Intracellular cytokine or responder cells. & added effector molecule producLower assay variability than tion (e.g., IFN-g, perforin) autologous assay when standard &Secretion of cytokines (e.g., responder cell batch is used. &Analysis at single cell level (flow IFN-g, IL-10) in supernatant cytometry). &T cell proliferation via &Simultaneous phenotypic MTS/MTT, 3[H]Thymicharacterization of stimulator and dine incorporation or CFSE responder cells possible (flow cytometry). dilution

Assay requires HLA-matched antigen- [27, 28, specific responder cells. 39, 40, &Intracellular protein production is not 42, 48] necessarily similar to secretion. & Effect of irradiation on DC functionality is not clear. &In vivo relevance of cell line as responder is not clear. &Analysis at population level (ELISA). &No simultaneous phenotypic characterisation of stimulator and responder cells possible (ELISA). &Cell source of secreted cytokines can not be determined. &Long assay (around 3 10 d). [17, 18, &Assay is based on allo-recognition (not 31, 35, MoA-related) instead of antigen speci41, 48, ficity. 67, 71, &Intracellular protein production is not 72]a necessarily similar to secretion. &Effect of cytokine addition on DC and T cell functionality is not clear. &Effect of irradiation on DC functionality is not clear. &Relatively high assay variability possible (i.e., T cell induction is depending on HLA mismatch). &Analysis at population level (except for flow cytometry). &No simultaneous phenotypic characterisation of stimulator and responder cells possible (except for flow cytometry). &Cell source of secreted cytokines can not be determined. (continued on next page)

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T cell stimulation assay (MLR)

IL-2 (when culture >7 d) &Nothing added

Key references

8 C. DE Wolf et al.

Table I (Continued)

Table I (Continued) Proposed in vitro potency assay

Stimulator cells

Responder cells

Additional Read-out parameter stimuli or cells

Tumor growth inhibition assay

bmDCs

Blood-derived T cells Adherent tumor line (after preincubation of DCs with T cells)

Inhibition of tumor cell proliferation (via change in electrical impedance)

Advantages

Limitations

Key references

&

&

[47, 75]

Antigen-specific functionality testing possible (in case autologous cells are used). &Combined assessment of DCmediated T cell activation and cytotoxicity. &Real-time monitoring of read-out parameter possible.

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Unless otherwise stated, DCs used as stimulator cells were mature (i.e., transduced to express tumor antigen, pulsed with tumor antigen or whole lysate and/or stimulated with a-specific maturation stimuli) APCs. bmDCs, bone marrow derived DCs (murine); BrdU, bromodeoxyuridine; CFSE, carboxyfluorescein succinimidyl ester; DC-SIGN, DC-specific ICAM-grabbing non-integrin (CD209); IP-10, IFN-g induced protein 10; MTS/MTT, tetrazolium salts; PCR, polymerase chain reaction; PD-L1, programmed death-ligand 1; TIL, tumor-infiltrating lymphocyte. a References are literature examples where these very similar assays are described. b This assay consists of two steps: transmigration of immature monocytes over endothelium (required for DC generation) and reverse transmigration (which reflects the DC migration also measured in other DC migration assays).

Regulatory perspective on potency assays for anti-tumour dendritic cells

Relatively long assay (around 3 4 d, differences between groups not visible before 2 d of culture). &Assay requires HLA-matched antigen-specific tumor cells. &Assay is only suitable for (strongly) adherent tumor target cells. &Assay can lead to overestimation of antigen-specific T cell function (e.g., in case of non-specific tumor cell loss). &Assay does not differentiate between indirect effects (via CTLs) and direct effects of DCs on tumor cells. &In vitro results may not correlate with clinical response in vivo. &Analysis at population level. & No simultaneous phenotypic characterisation of stimulator, responder and tumor target cells possible.

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should be able to migrate toward lymphoid tissue for T cell activation [29,35]. For most optimal antitumor responses, DC-mediated priming and activation of both helper CD4+ and cytotoxic CD8+ T cells and efficient cross-talk with NK cells are required [33,41,46]. The final aim of an anti-tumor DC-based therapy is reduction of tumor load via the induction of a strong antigen-specific immune response. To accomplish this, DCs should take up tumor antigens, differentiate into mature APCs and migrate to the lymph nodes [13,29]. There DCs have to activate antigen-specific T cells via contact between their MHC-peptide complex and the T cell receptor (TCR), co-stimulatory molecules and cytokines [7,13,29]. The final step involves migration to and recognition and killing of tumor cells by DCactivated T cells [13]. For adequate control of potency of a DC-based medicinal product, these aspects should all get attention during product characterisation and/or release. However, manufacturers often propose determination of only DC viability and phenotype as potency assay (for release). But that is generally not sufficient, because these parameters do not provide information about the actual DC functionality, unless expression of phenotypic markers is sufficiently linked to (in vivo) DC biological activity. To provide a broad overview of potential parameters to test for in an in vitro potency assay, we will focus in the next sections on assays specifically designed for the individual steps in the development of an immune response. Antigen uptake A first important aspect in the potency assessment of DCs is their ability to take up antigens. Development of an in vitro assay to test for the antigen uptake ability of DCs could be relatively easy because it will usually only involve the DCs to be tested and (labelled) antigen without the need for co-culture approaches. Several examples of antigen uptake (i.e., phagocytosis) assays using zymosan or dextran as a surrogate antigen have been described [23,31,41,47]. The relevance of these glucans as surrogate for the uptake of tumor antigens (usually proteins or peptides) will require attention. Use of reference proteins (like ovalbumin) may better reflect tumor antigen uptake, but use of actual antigens would be most appropriate. Because DCs are usually ex vivo-exposed to tumor cell lysate or loaded with tumor-associated antigens, control of antigen uptake is expected as part of the process and product characterisation (Figure 1)rather than of the product release testing, especially if antigen uptake is warranted by antigen

presentation (see section “Antigen presentation and co-stimulation”). If appropriate, an in-process control test for adequate antigen uptake may need to be implemented. When next to antigen(s) also messenger RNA (mRNA) (e.g., encoding for cytokines or co-stimulatory molecules) is added, its uptake and translation into protein (including further effects on DC functionality) should also be part of the process and product characterisation. Maturation Maturation is one of the key steps in the manufacturing process of a DC-based product; the cells will acquire a different phenotype and switch their functional profile from phagocytic scavenging to antigenpresenting [13]. The type of maturation stimulus (partly) determines the ability of DCs to induce T cell activation and polarisation. The only DC-based immunotherapy that has obtained a manufacturing authorisation in Europe so far is Provenge (sipuleucel-T; withdrawn in 2015). The potency assay of this drug product was based on a surrogate marker for APC activation: CD54 expression. Sheikh and Jones showed that quantitation of CD54 expression could be an appropriate assay for potency testing of this autologous DCbased immunotherapy because CD54 expression on the APCs (which were not specifically defined as DCs) was correlated with antigen uptake, processing and presentation [48]. This expression was also linked to co-stimulatory enhancement, which was assayed via a mixed lymphocyte reaction (MLR) [48]. In a follow-up study, overall survival appeared to be related to CD54 expression on the APCs, but this correlation was not strong [49,50]. Although these data support the use of CD54 as a marker for potency, both studies did not determine the type of T cells that were activated by CD54-expressing APCs. Furthermore, a link with in vitro T cell functionality was not established. The use of a CD54 blocking antibody in the co-culture between APCs and T cells could further support the suggestion that CD54 expression is a relevant surrogate marker for their ability to induce T cell activation. Taken together, CD54 expression is an acceptable potency criterion for Provenge batches, but (stronger) correlation with T cell functionality and in vivo clinical outcome would further confirm the suitability of CD54 as surrogate marker. Other maturation assays focus on the production or secretion of soluble mediators. Butterfield et al. described a quantitative assay that measured the ability of DCs to produce IL-12p70 because IL-12 is only produced by mature DCs and is thought to be of great importance for Th1 polarisation and

ARTICLE IN PRESS Regulatory perspective on potency assays for anti-tumour dendritic cells induction of anti-tumor T cells [32,51]. Already 1 day after co-culture between DCs and a CD40Lexpressing cell line (to mimic Th cell-like signals [52]), the cytokine level in supernatant was detected via an enzyme-linked immunosorbent assay (ELISA). Another advantage of this assay is the potential discrimination between different DC types that produce various levels of IL-12, which may be interesting for product characterisation purposes. In addition, this rapid assay may be useful as a release test, because CD40-CD40L interactions are important in DC activation and the use of a cell line will reduce assay variability [52,53]. However, comparison with primary human immune cells may be needed to demonstrate that the stimulator cells (in this case murine CD40L-expressing B cells) are sufficiently representative for the in vivo situation. In addition, it should be shown that changes in IL-12p70 production are linked to the DC capacity to stimulate the required T cell types, because some published data suggest that DCs producing no or only traces of IL-12 can still induce potent CTL responses [35,54]. And although IL-12 appears to be essential for CD8+ T cell and Th1 cell expansion, this cytokine may negatively impact the differentiation into (anti-tumor) memory cells, while induction of memory is part of the mechanism of action (MoA) of DC-based immunotherapeutics [33]. Another maturation assay based on a surrogate marker was proposed by Cornforth et al. [55]. The authors stated that C-C motif chemokine ligand (CCL) 17 serum levels were predictive for survival of patients treated with DC-based (GM-CSF adjuvanted) immunotherapy. An increase in CCL17 serum concentration of patient with melanoma between week 0 and 4 was associated with improved overall survival. In addition, a high CCL17 concentration at week 4 was associated with progression-free survival. Additional in vitro data could further support that CCL17 acts as an indicator of DC biological activity. This chemokine is a delicate example of a surrogate marker, but cannot directly be extrapolated to other products. In addition, CCL17 may not only be produced by product-derived cells but also by tumor-associated cells. In that case, CCL17 production is responsible for recruitment of Tregs, which is detrimental for an appropriate anti-tumor response [56,57]. This indicates that thorough investigation of the in vitro and in vivo surrogate marker source and the link between marker expression and clinical response will be required per individual medicinal product. Generally, maturation is tested during release via identity and purity parameters (phenotype). Insufficiently matured cells would, therefore, not be released for patient administration. From a regulatory point of view, determination of the DC phenotype for functionality purposes belongs to

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characterisation and not to release (Figure 1), unless a positive correlation between the surrogate marker (s) and the in vivo response has been shown, for which we have given two examples [49,55]. When clinical experience is too limited, using a surrogate marker may also be justified with results from an in vitro functionality assay performed during characterisation, for example, the relation of high IL-12 secretion by DCs with in vitro induction of functional antigen-specific CTL and Th1 responses [51,58,59]. Nevertheless, post-registration additional clinical data should be obtained to evaluate the link with the in vitro potency read-out. Migration Although migration of DCs from the site of injection to the lymphoid tissue is an essential step in activating an adaptive immune response, this phase is often overlooked in product characterisation [60]. Sufficient (non-)clinical effect is regarded as evidence for proper migration and T cell activation. Nevertheless, in vitro testing of migration may be useful to understand the behavior of the product in vivo and, for instance, justify its route of administration. Eyrich et al. proposed to test DC migration capacity in a transwell system [35]. This type of migration assay is potentially useful to determine the DC response to chemokine gradients as a mimic of active movement toward lymphoid tissue [35,61]. When an in vitro migration assay is used to characterise or control the potency of DCs, it should be shown that migration is not random but directed movement of DCs to the highest concentration of chemokines (e.g., CCL21). In addition, DC migration should be active, meaning that (i) the pore size of the transwell membrane should be small enough to prevent DC movements due to gravitational force, and (ii) in the absence of a chemokine gradient no or significantly less migration takes place. Also, determination of the phenotype of the migrated cells is essential to show that mature DCs are the main migratory cells and not cellular impurities like monocytes. Finally, data from Hansen et al., showing that autocrine CCL19 production by DCs interferes with migration toward a CCL21 gradient, indicate that interference of autocrine factors in the migration assay should be ruled out [62]. A more complex technique than the standard transwell system was developed by Higbee et al. [63]. Their in vitro Modular IMmune In vitro Construct (MIMIC) system included an assay to test for transmigration of cells over a layer of endothelial cells. Because this assay is part of a multi-tiered approach, this system will be further discussed in the section “Antigen-specific T cell activation and polarisation”.

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Evaluating migration capacity should be considered as part of product characterisation (Figure 1) and may be combined with other potency assays for product release. In case a specification for migration capacity is set (e.g., fold increase in specific migration compared with spontaneous migration), the in vivo relevance of the proposed limits should be justified. In addition, phenotypic markers such as C-C motif chemokine receptor (CCR)7 expression on DCs may already give an indication of the cellular migration capability toward a specific chemokine gradient [60]. It is, therefore, that DCs expressing a low amount of CCR7 may be intranodally injected instead of the skin or peripheral blood [64]. Antigen presentation and co-stimulation Once DCs have migrated toward lymphoid tissue, they will have to present their antigen to T cells. Few studies focus on this individual feature within the stimulation cascade of the adaptive immune response. Nevertheless, the level of the MHC-peptide complex on APCs largely influences T cell activation and even correlates with CTL responses to viruses and tumors [65]. Therefore, Neethling et al. proposed to evaluate the presentation and density of processed peptides in the context of MHC on the surface of DCs to assess the in vitro potency of DCbased products [65]. They introduced specific antibodies (TCRmimics) generated against the peptides loaded on DCs to confirm the presence of specific MHC-peptide complexes, even at low densities. This demonstrates that DCs had processed and presented peptides. In addition, it was shown that TCRmimics could specifically inhibit IFN-g production by CTLs in a DC-T cell co-culture, indicating MHC-peptide complex recognition by T cells. This detection and quantification of MHC-peptide complexes on DCs was proposed as surrogate marker for antigen-specific CTL responses. Although TCRmimics provide an interesting approach to characterise DCs, it may not be suitable as a release potency assay, because it requires specific antibody production for each product batch (i.e., patient-specific), making this assay very labor-intensive and costly. Antigen presentation alone is not sufficient for T cell stimulation. Co-stimulation (e.g., via binding of DC-derived CD80/CD86 to CD28 on T cells) and soluble factor production by DCs could significantly alter the expected T cell response. Appropriate characterisation and/or control of these parameters is also required. Shankar et al. developed an assay that could specifically test for the DC co-stimulatory ability instead of their antigen presentation capacity [66]. This socalled COSTIM assay makes use of a standard

allogeneic T cell donor, to lower the variability of the test, which is then co-cultured with the DC drug product and a low amount of anti-CD3 (for activation of the TCR) before T cell proliferation is analyzed. Because the assay only takes 2 days for completion, the allo-antigenic response is regarded as insignificant. Therefore, the COSTIM assay mainly tests for the function of co-stimulatory molecules on DCs, like CD54, CD80 and CD86. The COSTIM assay could be very informative for different DC products and could be easily combined with, for example, determination of cytokine production to broaden the (surrogate) functionality analysis [67]. Nevertheless, the authors recognised that their COSTIM assay should be regarded as quasi-quantitative because it is unknown what level of co-stimulation is physiologically relevant [67]. And since the measured T cell proliferation is not directly correlated with the specific antigens presented, the biological relevance of this assay needs further investigation. For the COSTIM assay to be acceptable as a release assay, an actual T cell functionality assay (during characterisation) should show that use of tumor antigen-loaded DCs will lead to T cells that are able to attack tumor cells expressing the specific antigen(s). Antigen-specific T cell stimulation and polarisation Based on the anticipated in vivo MoA, only antigenspecific induction of T cells is regarded as true DC functionality and should thus be shown during release (Figure 1). This requires a co-culture between DCs and responder cells (i.e., PBMCs or T cells) where the DC capacities antigen presentation, co-stimulation and polarisation are simultaneously analyzed. According to Shankar et al., the ideal potency assay for DC-based therapies would evaluate the capacity of antigen-loaded DCs to stimulate autologous antigen-specific T cells [66]. In line with this, Eyrich et al. developed a T cell priming assay that was specifically based on CTL activation [35]. Na€ıve CD8+ T cells from healthy donors were cultured with autologous peptide-pulsed DCs and a cytokine mix. Induction of antigen-specific CTLs and production of IFN-g after re-challenge with an antigen-specific tumor cell line was used as indicator of potent T cell priming. The data suggest that this assay is capable of measuring the induction of a functional in vitro anti-tumor response, although it remains to be demonstrated that CTL cytokine production after interaction with tumor cells is correlated with antigen-specific tumor lysis. However, the assay should also be qualified with cells from patients, which may have a different immunologic history, which impacts the anti-tumor response [33].

ARTICLE IN PRESS Regulatory perspective on potency assays for anti-tumour dendritic cells Studies by Wei et al. and Schnurr et al. describe a co-culture assay between moDCs pulsed with tumor cells or lysate and autologous PBMCs [41,68]. Weekly re-stimulation with fresh DCs and several cytokines was required to stimulate antigen-specific T cells. After several rounds of re-stimulation, the T cell proliferation, marker expression and cytokine production were determined. Cytokine production was analyzed either in the culture supernatant [41] or intracellularly using flow cytometric analysis [68]. The latter approach would be preferred, because it provides information on the cytokine cell source [12]. As expected in an autologous system, individual donor results showed large variability in T cell proliferation. In addition, upregulation of CD69 expression on T cells [41] was only marginal, but this increase may still be of significance regarding the antigen-specific nature of these cells. Both studies showed a positive correlation between the read-outs and the in vitro cytotoxic response, although actual correlation with the in vivo response still has to be determined. All above-mentioned studies made use of autologous cells, therefore, these assays may provide a good reflection of the anti-tumor response in the patients. However, practical considerations may limit their use. These assays require a relative large amount of DCs (from precious and limited drug product) and responder cells. Also, the duration of the cultures prevents rapid availability of potency results. In addition, assay variability is high due to the use of autologous cells and additional variation may be induced with each round of re-stimulation. Further assay optimisation, such as using only one round of stimulation or another short setup, would, therefore, be required before an autologous assay could be suitable for release. A rapid autologous cell-based assay was described by Lin et al. [38]. They cultured tumor antigen-transfected mature DCs with autologous T cells for only 3 days and subsequently determined T cell proliferation. Unfortunately, high amounts of IL-2 and mitogen phytohemagglutinin (PHA) were added, which abolished the antigen-specific character of the assay. In addition, proliferation was determined via a colorimetric method based on the cellular metabolic activity at the end of the culture, which may not necessarily reflect the metabolic state during the culture. Moreover, differences in metabolic activity not all directly relate to changes in proliferation [69,70]. Thus, despite the advantage of a short assay duration, the use of this assay to characterise and control the capacity of DC products to induce antigenspecific T cells is questionable. Another autologous culture approach (which takes 1 to 2 weeks to perform) is the commercially available high-throughput in vitro MIMIC system,

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which was already briefly mentioned as a migration assay. This MIMIC system is originally developed to reflect parts of the complex human in vivo immune response after preventive vaccination. This would allow for reduction in the use of animal models and for improvement of the relevance of the results for the human situation (by making use of individual donor cells) [63]. In the MIMIC system, the peripheral tissue equivalent mimics DC maturation and transmigration through endothelium to reach lymph nodes. The lymphoid tissue equivalent detects crosstalk between DCs and T or B cells in an artificial lymph node milieu. Additional assays can subsequently be used to assess the function of cells stimulated by DCs (e.g., cytotoxicity testing). The advantage of this MIMIC system is that a proper overview of individual immune reactions is generated via the simulation of several in vivo situations relevant for induction of an immune response. In addition, the influence of changes in DC culture conditions or the presence of co-medication can be analyzed. The MIMIC system appears to be a very sensitive method in the determination of, among others, cytokine responses. However, for a release potency assay, a correlation between the read-out of the lymphoid tissue equivalent assays and T cell cytotoxicity should be demonstrated, or the MIMIC system should be combined with an actual T cell functionality assay. In addition, as with every other in vitro assay, all cell ratios and concentrations used in the tissue equivalent assays will require support from data obtained with characterisation and nonclinical studies. To overcome the limitation of long culture periods in autologous culture settings, one can use human leukocyte antigen (HLA) matched T cell lines as responder population [39,40,42]. These assays usually take only a few days to perform and analyze DC functionality in an antigen-specific manner. For ‘off-the-shelf’ medicinal products consisting of a DC line (e.g., pDC line pulsed with tumor antigens or myDCs endogenously expressing leukemiaassociated antigens [27,28,71]), use of HLAmatched instead of patient-derived responder cells is even inevitable to detect antigen-specific T cell stimulation. Nevertheless, development of HLAmatched cell clones for each individual patient is costly and time-consuming and it is, therefore, expected that such assays will be suitable for characterisation purposes only and not for product release testing. For DC products consisting of allogeneic cells, a MLR may be regarded as a useful alternative for HLA matching [17,18,31,35,41,48,71,72]. However, a MLR is also often proposed as a release test for autologous DC products. Although some

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consider a MLR the golden standard for DC potency, because these cells are such potent inducers of T cells [73], this approach is not preferred. Testing for the allo-stimulatory capacity of DCs is not representative for DC activity in the context of autologous interactions and allo-recognition does not detect antigen-specific T cell stimulation, the actual mode of action. However, development of an antigen-specific release assay for potency may not always be feasible. In these cases, a MLR for release could be considered, provided that during characterisation of clinical batches a correlation with antigen-specific functionality assays is shown. In addition, the assay should be optimised to reduce the impact of differences in allo-antigens between (both effector and responder) cell batches on assay outcome, for example, by implementing appropriate assay controls and the use of standardised responder cell batches. Besides T cell proliferation, functional antigen presentation via MHC-peptide-TCR interactions will also induce cytokine production by DCs (and T cells). Therefore, polarisation of responder cells (i.e., frequency of T cell subsets) may be a read-out for DC potency. A potent DC product should induce tumor-rejecting Th1 and Th17 cells via IL-12 and IL-1b production, while not inducing Th2 and suppressive cells via IL-10, transforming growth factor-b and (indirectly) IL-4 production [51,74]. However, in vivo cellular interactions are much more complex and are not necessarily reflected by in vitro polarisation. For example, in vivo localisation of T cells is expected to impact their phenotype and function. Therefore, DC production of T cell polarising cytokines alone is considered insufficient as a functionality determinant and should be regarded as a surrogate marker for DC functionality. Correlation between in vitro cytokine levels and in vivo clinical responses will be required to show the relevance and suitability of such a surrogate marker. In contrast, DC-mediated cytokine production by T cells is regarded as an appropriate parameter for DC functionality (at release testing), although a relationship between cytokine production and cytotoxicity should be shown during characterisation, as discussed below. T cell functionality As a quality control test for release, it is considered sufficient to show that DCs are able to directly induce T cell activation or harbor specific surrogate markers that are linked to such T cell activation. Nevertheless, characterisation studies would be expected to include a demonstration of tumor recognition and attacking capabilities by the activated T

cells (Figure 1). Obviously, the ideal functionality assay would be based on killing of (patient-derived) tumor cells by autologous T cells [12]. However, such an assay is expected to be impractical and insufficiently robust. Therefore, assays using suitable surrogate cells (e.g., tumor cell lines) may be developed, provided that the cells are relevant for all patients, regardless of their HLA type. In addition, it may be useful to test for antigen-specific T cell functionality on more than one (surrogate) cell type, such as tumors with high and low expression of the antigen as performed by Lin et al. [38]. Previously, we have discussed numerous aspects of potency assay principles and techniques and their suitability to test anti-tumor functionality of (DCactivated) T cells [12]. Usually, T cell functionality is determined in an assay separate from the co-culture with product-derived DCs. Few studies determine T cell cytotoxic potential in the presence of the DCs that activated these T cells. Pham et al. determined the effects of a mixture of murine DCs and DCinduced T cells on tumor target cells in real time, using the commercially available xCELLigence system [47,75]. Significant inhibition of adherent tumor cell proliferation compared with controls could be measured as a change in electrical resistance (after 30 h of culture). However, because allogeneic T cells were used, mismatch responses could (at least in part) be responsible for their activation by DCs, which greatly impacts the relevance for DC medicinal products. Nevertheless, the principle of this assay could be used for human autologous cells and HLAmatched tumor cells. Another concern is that the assay read-out is not cytotoxicity but inhibition of proliferation and non-antigen-specific tumor growth inhibition (e.g., via enzyme release from dying CTLs) can not be excluded. Selection and design of potency assays In vitro potency assay(s) for release of DC medicinal products should reflect one or more of the cells’ relevant in vivo functions [45]. However, all individual features from antigen uptake and subsequent maturation to the activation of the right responder cell (sub)population contribute to DC potency (and thus clinical outcome). Therefore, these aspects should at least be covered during characterisation (Figure 1). For release testing, choosing the right assay(s) and (surrogate) parameters to test for DC functionality seems to be critical. This choice should be productspecific, because differences in DC source, production methods (including isolation, antigen-loading and maturation), route of administration and vaccination schedules have been shown to impact the results of in vitro testing and/or the clinical response.

ARTICLE IN PRESS Regulatory perspective on potency assays for anti-tumour dendritic cells Nevertheless, to provide some cell-specific guidance for in vitro potency assays, we have discussed advantages and limitations of frequently proposed DCbased functionality assays and included remarks concerning additional characterisation. We will end this review with some specific points-to-consider during the selection and design of an in vitro potency assay. Choice of a potency assay The ideal in vitro potency assay for release is based on a co-culture of (mature) DCs with autologous responder cells to determine the activation of antigen-specific cells. However, for release testing of most products this will not be feasible because this requires a long culture period (with increasing interdonor variability) and a large amount of drug product (limited resource). Nevertheless, the use of autologous assays in characterisation studies (including confirmation of the actual functionality of the responder cells) and the search for a suitable release assay based on the MoA are strongly encouraged. A MLR is not the preferred approach, because the anticipated MoA for DC products for anti-tumor treatment presumes the antigen-specific induction of other immune cells. Read-out parameters With respect to co-culture assays aimed to detect T cell stimulation, both T cell proliferation and T cell cytokine production may be used as a marker for DC potency. The method to determine cytokine production levels should preferably also address the cell source to confirm that changes in cytokine production are due to T cell induction. Other indications of T cell activation and modulation by DCs (changes in avidity of CTLs for MHC class I molecules on tumor cells, expression of factors regulating immune checkpoints or CTL migration into tumors, etc.) may also be considered, but are not generally evaluated and their clinical relevance requires further investigation [33]. In any case, the choice of assay read-out should be justified by data, linking it to the intended T cell function (i.e., tumor cell lysis) [12]. In addition, correlation with in vivo effects (like tumor load reduction or improved patient survival) is expected. In case in vitro induction of antigen-specific responder cells can not be used as a potency marker for release, surrogate markers for DC functionality could be considered. Cell surface markers related to DC maturation and production of cytokines that are able to contribute to responder cell induction are generally considered the most suitable surrogate potency markers. The degree of DC maturation is usually tested as a difference in expression of surface

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markers (like MHC II, CD80 and CD86) and chemokine receptors (mainly CCR7). Determination of in vitro produced cytokines related to T cell induction mainly involve IL-12p40 or IL-12p70. However, in vivo cellular communication is considerably more complex and is not necessarily reflected by the in vitro cytokine production. When such surrogate markers are proposed for release, their relevance and specificity for DC functionality should be justified with product-specific characterisation and (non-) clinical data that are ideally linked to marker expression/production levels. In addition, a quantitative specification (e.g., % positive cells, % cells with a signal above a relevant threshold or fold increase in % marker expression per cell) would be preferred, especially when a parameter is expressed both on immature and mature cells. Comparing gene expression profiles of different starting materials, product intermediates or final products might help to identify and select the most relevant parameters to assess one or more DC functions [76]. Also, similarities and differences between healthy donor material (which is used for development or validation of potency assays) and patient material could be investigated by gene profiling. Designing rapid tests that can reliably detect functional expression of these genes may then lead to development of a suitable assay matrix for release. The correlation of these parameters with in vivo functionality and their clinical relevance should, however, still be shown. Co-culture assay optimisation Assay design and optimisation is probably most challenging for assays based on co-cultures between DCs and responder cells. Some considerations for the culture conditions are provided here, as an antigen-specific co-culture assay is likely to play an important role in product characterisation and justification of the choice of surrogate potency markers. Responder cell types Induction of both CD8+ and CD4+ T cells responses with abolition of suppressor cell responses (e.g., Tregs) and breakdown of the immunosuppressive tumor microenvironment are key factors for an efficacious anti-tumor therapy [3,5,30,33]. However, generally, most potency assays do not differentiate between CD8+ and CD4+ T cells (after a co-culture between DCs and CD3+ T cells), even when DCs are loaded with a combination of MHC class I and II peptides [34]. Because the induction of functional multi-cell types is considered part of the anti-tumor DCs’ MoA, the presence and functionality of T cell subsets and their specific contribution to the

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anti-tumor response should at least be investigated in characterisation or (non-)clinical studies [33]. This should also include use of (autologous) responder cells from patients because their immune history might differ from healthy donors. These data can substantiate the choice for a specific responder cell (sub)population in the release assay. Apart from T cells, other immune cell types also play crucial roles in the regulation of an anti-tumor response [33]. Especially NK cells would require attention in immunomonitoring, because these cells and DCs positively influence each other at sites of inflammation [7,9]. Studies in mice have shown that functional NK cells are important for tumor elimination [77]. They can also directly help tumor-specific T cells, for example, via their cytotoxic capacity [20]. For specific products, DC-mediated NK responses might even correlate more closely with the clinical outcome than T cell responses [78]. Thus, in characterisation studies, also the contribution of tumor cell lysis by NK cells, both in the in vitro potency assay (where NK cells may be present in the cell culture) and with respect to the in vivo MoA, should be considered. Culture duration Co-culturing pro-inflammatory DCs with T cells for only a few hours may show higher Th1 activation compared with a longer co-culture [18]. DCs continue their maturation (even when a maturation stimulus is no longer present) and finally turn into anti-inflammatory APCs, with a negative impact on T cell activation or even the formation of Treg-like cells. This property of DCs is essential for in vivo fine-tuning of the immune response and preventing overstimulation of effector T cells. However, in vitro where mature DCs and responder cells are usually co-cultured for several days this may impact the outcome of the functionality assay. Therefore, proper assay controls are needed. Assay characterisation studies may be required to determine the optimal moment of DC addition and duration of co-culture with responder cells. Cell ratio Apart from the duration of DC and T cell interaction, the ratio between these cells is important. This ratio appears to be quite variable among different studies. For some co-cultures different ratios were used, showing a ratio-dependent T cell response [17,35,41,47,48,66,67,74,75]. Such studies could be valuable for product characterisation, for example, to prevent misinterpretation or rejection of a useful potency assay. The optimal and physiologically relevant ratio between stimulator and responder cells for the release potency assay should be determined. When determining this ratio, it should be taken into

account that a specific DC:CD3+ T cell ratio may be optimal for CD8+ T cells, but not for CD4+ T cells or vice versa. Exogenous stimuli In general, no additional factors other than the maturation stimulus for DCs or a low amount of cytokines for T cells are added to optimise the response. If relevant, it should be justified that addition of exogenous stimuli to the culture will not abolish the antigenspecific character of the assay. In some cases, potency assays should specifically address the impact of adjuvants (such as GM-CSF) on DC functionality, for example, when adjuvants are used in parallel with the cell-based treatment or when they are part of the final product [3,5,55]. Link with the in vivo response In the previous sections, we highlighted that, for a proper justification of the selected potency assay(s) for control of a DC product, the relevance of the in vitro assay for the clinical response should be demonstrated. In vivo induction of antigen-specific T cell responses could be evaluated via immunomonitoring, for example, by in vitro tumor antigen re-stimulation of PBMCs or T cells derived from patients at different time points before, during and after immunotherapy [79]. A good example of this immunomonitoring approach is found in a clinical study from Carreno et al., where PBMCs were analyzed weekly to define the presence and cytotoxic potential of antigen-specific T cells (after in vitro re-stimulation with tumor antigens) [51]. Although time-consuming, this may be a relatively straightforward method to correlate in vitro functionality of the DC product to the actual in vivo clinical effect on T cells. However, cellular reactions measured in peripheral blood do not necessarily reflect the overall anti-tumor response. Therefore, it will also be important to evaluate whether the in vitro potency can be linked to the clinical outcome (such as tumor reduction or patient survival) and whether the potency assay is capable of distinguishing potent and sub-potent product batches. The few published studies that determined the relevance of a potency assay type or read-out primarily focussed on surrogate potency markers, such as surface receptor expression or effector molecule production [49,51,55]. There are also examples where a comparable in vitro assay may be suitable to test the functionality of one DC product, but not of another. For a DC product against papilloma-related cancer, in vitro DC-mediated T cell activation reflected the in vivo response [39,40], in contrast to a DC product against melanoma [74]. This

ARTICLE IN PRESS Regulatory perspective on potency assays for anti-tumour dendritic cells exemplifies that the in vitro potency approach should be established per individual DC product. We noted in scientific advice requests that for the majority of DC products the correlation with the clinical response had not been assessed, probably because of limited clinical experience. If a link with clinical outcome can not be evaluated, a combined approach with analysis of effects on responder cells at tissue level (tumor location or draining lymph nodes) and non-clinical evaluation may be required to demonstrate the link between in vitro and in vivo outcomes and/or identify additional potency-related parameters. For example, Kellermann et al. and Vissers et al. found that several tissue-specific and in vitro cultured DC types showed a comparable migration response or expressed comparable T cell attracting chemokines [16,61]. This suggests that chemokines or chemokine receptors could be physiologically relevant surrogate markers for DC functionality. From experience we know that it may not be feasible to demonstrate a link between the in vitro potency measured at product release and the clinical outcome. In that case, the relevance of the in vitro potency assay should be justified based on the overall approach used for potency characterisation and control of the DC product. Future perspectives and conclusion Regardless of their indication, individual DC drug products differ in their source of origin and manufacturing methods. These differences in DC products will result in differences in the characteristics of stimulated T cells with regard to proliferative, memory, homing and other functional capacities. Therefore, each DC product is considered unique and requires its own control strategy to ensure consistent quality and potency. This means that all potency assays will be assessed on a case-by-case basis, thereby not precluding any functionality test in advance. In this review, we have evaluated assays designed to test individual or combined DC functions (Figure 1). These functions should be warranted in characterisation studies. At least induction of (antigen-specific) responder cells should be shown during release, because this DC function directly relates to the anticipated in vivo MoA. A surrogate marker (or markers) for this responder cell induction may also be appropriate, provided that such marker is linked to the in vivo DC biological activity and clinical outcome. The focus of this review has been on DC products used in anti-tumor therapy. Nevertheless, proinflammatory DCs may also be used to boost

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immune responses against chronic viral infections, such as those caused by HIV or hepatitis B virus [80,81]. Likewise, there is a growing field of tolerogenic DC-based immunotherapy in auto-immunity and transplantation settings (e.g., [82 85]). In contrast to DCs in pro-inflammatory treatments, these DC-based therapies should result in re-establishment of immune tolerance, for example, by induction or restoration of functional Tregs. Several studies regarding auto-immune diseases have shown that presentation of immunogenic self-antigens by tolerogenic DCs may be able to induce protective regulatory cells (e.g., [86 89]). Also, development of more standardised tolerogenic DC drug products has recently been proposed [90]. Accordingly, the search for adequate potency markers and assays to characterise and control DCs products will continue. During the last decades, many studies have increased our acquaintance with the complex family of surface marker defined DC subsets and their unique features in the immune response. Future investigations are expected to further elucidate the extensive involvement of DCs under both healthy and pathological conditions. Consequently, the DC subsets required for specific therapeutic indications may be better defined and manufacturing processes improved accordingly to yield the desired DC population. To be able to substantiate the quality of these DC products, development of relevant in vitro potency assays and guidance is expected to ensue.

Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial or notfor-profit sectors. The figure was produced by using Servier Medical Art (https://smart.servier.com/). Disclosure of interests: The authors have no commercial, proprietary or financial interest in the products described in this article. The contents of the article represent the authors’ personal opinion and do not necessarily reflect any position of the Dutch Medicines Evaluation Board.

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