Background
Regulatory T (T
reg) cells are an attractive type of advanced therapy medicinal product (ATMP) for adoptive cell therapy that can be used when the restoration of immunotolerance to self- or allo-antigens may prevent or even cure diseases [
1‐
3].In murine models, expanded T
reg cells have been shown to be effective for the induction of long-term tolerance to bone marrow transplantation, for the prevention of graft-versus-host disease (GvHD), and for prolonging heart and skin allograft survival [
4‐
7]. In humans receiving human leukocyte antigen-haploidentical hematopoietic stem cell transplantation for various malignancies, T
reg cell adoptive transfer prevents GvHD without reducing the graft-versus-leukemia effect [
8]. At the time of writing the present article, the safety and the potential clinical efficacy of ex vivo-expanded autologous polyclonal T
reg cells are under evaluation in 48 clinical trials worldwide for indications such as end-stage kidney disease (KD), kidney or liver transplantation, type 1 diabetes, and GvHD [
9]. In these conditions, T
reg cells could be a promising therapeutic tool to promote donor-specific transplant tolerance by exerting their immunomodulatory properties in controlling allograft rejection, for both therapeutic and preconditioning regimens, thus possibly allowing reduction and/or discontinuation of immunosuppressive drugs [
10].
A critical topic for clinical applications is whether to expand T
reg cells from an autologous or allogeneic source. The main issue in using the latter is the risk of rejection and the resulting short survival of the donor cells, as well as possible alloimmune sensitization [
11], whereas the major challenge related to an autologous product might be the difficulty in expanding T
reg cells and thus achieving the therapeutic dose, due to the patient’s pathology [
12]. Furthermore, the manufacturing costs of an autologous product are higher than those of an off-the-shelf allogeneic product, since each batch is patient-specific [
13]. However, while both autologous and allogeneic T
reg cells have been used in hematopoietic stem cell transplantation [
12,
14], autologous cells are the preferred choice in solid organ transplantation [
15].
Evidence from preclinical models suggests that the ratio between T
reg cells and T effector (T
eff) cells needed to promote tolerance to organ transplantation should be much higher than the physiological level [
16,
17]. Indeed, in a normal peripheral blood sample, the frequency of circulating T
reg cells remains constant and low (representing 2–8% of CD4
+ T cells, < 2% leukocytes [
15,
18,
19]), and a therapeutic number of T
reg cells can only be achieved following their in vivo or ex vivo expansion [
17,
18,
20]. Several expansion protocols have been proposed to obtain a pure T
reg cell population that can retain its suppressive function [
15,
20‐
23]. In general, an effective expansion protocol includes cultivation for 3–4 weeks in the presence of anti-CD3/CD28 beads, interleukin (IL)-2, and rapamycin [
24,
25] to ensure a 20–200-fold increase in the number of T
reg cells without impairing their immunoregulatory activity [
20].
In the European regulatory framework, T
reg cells enriched by immunoselection are not considered as a medicinal product and are regulated under the European Union Tissue and Cells Directive 2004/23/EC [
26]. Instead, T
reg cells expanded ex vivo are classified as an ATMP, which is a substantially manipulated cellular product according to the definition in Article 2 of Regulation (EC) N. 1394/2007 of the European Parliament and of the Council of November 13, 2007 [
26]. This means that expanded T
reg cells must be authorized by national competent regulatory authorities to be used in a clinical trial and must be approved by the European Medicines Agency to be marketed.
As part of our collaborative interinstitutional ATMP development program for adoptive cell therapy in organ transplantation, in the present paper, we describe in detail some practical issues of the whole process for T
reg cell expansion, starting from Good Manufacturing Practice (GMP) validation; in particular, the novel items are discussed [
18,
20,
27‐
35]. We also pay special attention to compliance with the most recent European regulatory guidelines concerning GMP for ATMPs [
36], which strongly affirm the crucial importance of a risk-based assessment to identify the potential risks associated with the manufacturing process and to control/mitigate them.
First, the practical approach we followed to design the validation process is illustrated, and then the assessment of its performance to produce GMP-compliant, clinical-scale ex vivo-expanded Treg cells from patients with end-stage liver disease (LD) or KD is described. In addition, quality control (QC) method validations are explained.
Discussion
Herein, we describe the process validation for a safe, reproducible, and flexible GMP manufacturing process for isolation, expansion, and cryopreservation of expanded Treg cells from patients affected by end-stage KD or LD.
The aim of this work was to describe the steps followed for the validation of a manufacturing process adaptable to small-size and/or academic pharmaceutical plants to obtain a final product available for clinical applications in which immunomodulation is required. This objective is particularly valuable when considering that Treg cells can positively affect or even counteract the evolution of severe diseases and/or reduce the need for immunosuppressive therapy, with beneficial results on therapy-related side effects and healthcare costs.
In a healthy human adult subject, T
reg cells represent up to 5–10% of the total circulating T cells [
3], meaning that at most 200 × 10
6 T
reg cells from leukapheresis can be isolated [
30]. Therefore, ex vivo expansion is essential to obtain a sufficient number of T
reg cells in order to impact the immune response to an allograft after transplantation in humans. In this regard, various groups have recently demonstrated a beneficial T
reg cell-dose dependent effect on alloreactivity suppression for tolerance induction after liver transplantation [
43,
44].
Due to the extent of cell manipulation, expanded T
reg cells are classified as an ATMP according to the European regulatory framework [
26]. As recommended by the European Medicines Agency regarding the inclusion of the quality-by-design approach for the development of an investigational medicinal product, we performed a process risk assessment in which all sources of variability potentially affecting a process are identified, explained, and managed by appropriate measures. For this purpose, we used PHA to identify, classify, and describe possible risks, dangerous situations and events that could cause failure, their origin, and possible consequences (risks) as well as to estimate the probability of occurrence for a given potential failure.
We set up a detailed strategy (Table
1) by which we could identify different potential failures according to the category (e.g., equipment, personnel, reagents, suppliers, environment, and the patient or the process itself) and their possible consequences, and we gave a severity score to each of them according to the respective severity of occurrence (i.e., a higher score indicated a higher risk). Mitigation strategies were proposed for each encountered risk, leading to a significant reduction of the possible final score. As mentioned, this method defines the corrective actions to modify, control, or delete dangerous situations as well as measures the safety and reliability of the manufacturing process. Furthermore, it could be of great use as a guide for the implementation of corrective actions from the very beginning (from the proper use of standard operating procedures, to the appropriate training of the personnel, traceability systems, etc.).
We also provided a practical example of how we managed an unexpected deviation that actually occurred for the transport of starting material. Indeed, despite the implementation of control strategies, a deviation could still happen. According to GMP, an investigation file must be immediately opened to classify the severity of the failure and to identify the causes, with the aim of preventing or limiting the possible negative impact to the process. The investigations and corrective actions implemented must be recorded in dedicated documentation that must become an addendum to the batch record and must be evaluated before the validation process is approved. Indeed, a possible deviation can always occur, not only in the validation phases of a process but also during a clinical trial, and it should be properly managed in time and with an appropriate approach according to GMP, rather than leading to a possible rejection of a clinical sample.
To monitor the progression of such a long manufacturing process, besides the identification of the critical quality attributes, establishment of the appropriate assays at different steps of the process to ensure the quality of intermediate and finished products is critical. In our opinion and based on our experience, even if the validation of a production process and the QC methods are only suggested in the last revision of the regulations concerning ATMPs [
36], even the most accurate risk analysis cannot completely replace a validation step, especially for the analytical methods. Therefore, the release QC validation should be performed according to the official Pharmacopoeia whenever possible (e.g., for compendial methods).
The results of the validation work for the different parameters indicated reliable results for viability and purity, with positive consequences on the robustness of our manufacturing process and on its ability to produce a high-quality ATMP.
A critical step to consider before clinical use is the depletion of immunomagnetic beads from the expansion product. The efficacy and safety of anti-CD3/CD28 expansion beads in vivo are actually not well known. To ensure safety of the finished product on day 21, bead removal from expanded T
reg cells was performed by magnetic immunodepletion, according to the manufacturer’s instructions. For QC of residual bead enumeration, we performed a specific validation in agreement with GMP, as the procedure proposed by the manufacturer is not intended for drug development but for research use only [
45].
Therefore, we set and validated a method for bead enumeration in the finished product based on the raw count in the Bürker chamber (see the “Materials and Methods” section for details) and the use of a reduced number of cells for QC, compared to what is required by the protocol proposed by the manufacturer. We decided not to follow the manufacturer-suggested procedure because of several critical issues: (1) most cytometers do not provide an absolute event count without the addition of counting beads, which was difficult in our case, since the reference beads were difficult to distinguish from the expansion beads; (2) the number of “wasted” cells (original fraction corresponding to 5–20 × 104 MACS GMP ExpAct Treg Beads and 1 × 108 cells for the target fraction, both in triplicate) required for bead enumeration affected the clinical dosage; (3) repeated centrifugations and discarding of the supernatant would invariably lead to unpredictable and unstandardized bead loss, and the consistency might vary depending on the different sample matrixes (e.g., original fraction, target fraction, and control), thus affecting the method accuracy.
Due to a robust and consistent expansion capability, regardless of the number of circulating Treg cells, the process we validated will allow patients to be enrolled in clinical trials. Indeed, we demonstrated that despite the fact that the cellularity of the apheresis product may vary due to the harvest procedure, the Treg cell content in the starting material does not influence the isolation efficiency.
With the manufacturing process we described, we were able to obtain a clinically relevant cell dose of 79 ± 23 × 10
6 T
reg cells/kg for a mean body weight of 70 kg; of note, a target cell dose for a clinical trial is generally 1–10 × 10
6 T
reg cells/kg [
3]. Moreover, ex vivo expansion also allowed us to obtain a purer product than that obtained by direct isolation. In our experience, large-scale T
reg cell selection using the CliniMACS isolation system from leukapheresis yielded a CD4
+ CD25
+ T-cell purity of 55% (range 42.6–62%), the majority of which expressed FoxP3, in keeping with the reported data from healthy subjects [
40,
46,
47].In response to recent studies showing the negative effect of cryopreservation on T
reg cell function [
41,
42], we have previously demonstrated that expanded T
reg cells after thawing can effectively prevent the onset of xenogeneic GvHD as well as improve acute GvHD and survival in a mouse model of GvHD using immunosuppressed mice (i.e., NOD-SCID-gamma knockout mice) [
7]. Herein, the in vitro data reported also confirmed our previous results for T
reg cell expansion according to our GMP-compliant process from patients with end-stage LD or KD.
Finally, the manufacturing process that we set up has the important aspect of flexibility, which might be extremely useful to comply with different logistic and clinical settings. A 21-day expansion process can be demanding for small academic groups like ours. For this reason, we examined the possibility of fractionating the expansion in order to have a process more adaptable to the needs of the laboratory. On this point, our preliminary data from a single run suggested that it might still be possible to restore the expansion ability of Treg cells after thawing the intermediate product (e.g., 14-day-expanding Treg cells). Indeed, according to the expansion curves we obtained, a high number of cells on day 14 was available to be frozen as a master cell bank for future expansion. Furthermore, starting from cells frozen at this point of the expansion curve would allow the more rapid achievement of a clinically relevant number of cells in only 7 days, with all the logistical advantages of a shorter time period and an easily programmable production facility. This means that the timing of infusion can be adapted to different conditioning schemes and even to occasional deviations due to logistic and/or clinical problems during a clinical trial. Also, it would give the clinician the opportunity to plan ATMP administration at the optimal time based on the patient’s clinical progress in the context of the adaptive study design.
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