key: cord-0800031-fmshmvxp
authors: Roemhild, Andy; Otto, Natalie Maureen; Moll, Guido; Abou-El-Enein, Mohamed; Kaiser, Daniel; Bold, Gantuja; Schachtner, Thomas; Choi, Mira; Oellinger, Robert; Landwehr-Kenzel, Sybille; Juerchott, Karsten; Sawitzki, Birgit; Giesler, Cordula; Sefrin, Anett; Beier, Carola; Wagner, Dimitrios Laurin; Schlickeiser, Stephan; Streitz, Mathias; Schmueck-Henneresse, Michael; Amini, Leila; Stervbo, Ulrik; Babel, Nina; Volk, Hans-Dieter; Reinke, Petra
title: Regulatory T cells for minimising immune suppression in kidney transplantation: phase I/IIa clinical trial
date: 2020-10-21
journal: BMJ
DOI: 10.1136/bmj.m3734
sha: 500c5e413273c392499dec05f5d83b4571172cc7
doc_id: 800031
cord_uid: fmshmvxp

OBJECTIVE: To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model. DESIGN: Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13). SETTING: Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union). PARTICIPANTS: Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9). INTERVENTIONS: CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×10(6) cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48. MAIN OUTCOME MEASURES: The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio. RESULTS: For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire. CONCLUSIONS: The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies. TRIAL REGISTRATION: NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

: Descriptions and phenotypes of immune cell populations. 20 Table S6C : Longitudinal analysis of absolute counts of leukocyte cell subsets. 21 Table S6D : Longitudinal analysis of leukocyte subset frequencies.

22 Table S6E : Longitudinal analysis of gene expression levels of whole blood samples. 23 Table S7 : Summary of the Patient Assessment during the Trial (Table continued on next page).  24  Table S7 : Summary of the Patient Assessment during the Trial (Table continued from previous page) . 25

The primary Clinical Endpoint was the incidence of biopsy-confirmed acute rejection (BCAR) within 60 weeks of organ transplantation. Histopathological grading of biopsy material was performed according to the Banff criteria (1). Patients with subclinical rejection detected by a protocol biopsy will not register a primary endpoint as long as the patient has a stable graft function with no signs of allograft rejection. The management of patients according to histopathological findings was done at the discretion of the responsible nephrologist. BCAR has been selected as the primary endpoint for the following reasons:

• The incidence of BCAR has become an accepted short-term surrogate marker for renal allograft survival and is widely used as a primary endpoint in renal transplantation trials of immunosuppressive therapies (2, 3).

• Allograft biopsy is the "gold-standard" for the evaluation of renal allograft dysfunction (4, 5) and the histopathological criteria for the diagnosis of acute rejection are standardized and unambiguous (1).

• BCAR represents the most pragmatic and testable primary endpoint for treatment efficacy within the time and resource constraints of a transplantation trial.

To assess BCAR, kidney graft biopsies were performed either as scheduled protocol biopsies, or as unscheduled for-cause biopsies. One protocol biopsy was mandatory at 36 weeks posttransplant (Visit 8) to guide the IS drug tapering. Additional, (optional) protocol biopsies were performed intermediately to identify early signs of graft pathologies. For-cause biopsies were performed in case an acute rejection episode was suspected. 

We aimed to get a global overview of the samples and the general sample quality. The number of clonotypes ( Figure S5A ) gave us an impression of the size of the obtained repertoires. First of all, we saw a large range from 23 to 100387 clonotypes. We next inspected the number of accepted reads across the pooled samples (Figure S5B) , and looked at the total number of Imseq accepted reads found in each sample in case the different runs are pooled. As a side-note, it is not surprising, if the total number of accepted reads is larger than the number of cells, because of the amplification process. For quality reasons, we removed samples with less than 20000 reads, which affected one sample of patient C5051 (V10 -17602 reads / 393 clonotypes) and two samples of patient C5057-R (V08 -4857 reads / 253 clonotypes and V10 -333 reads / 23 clonotypes), while the other n=44 samples met our set quality criteria, thus only loosing 6% of our samples for analysis due to technical reasons.

We give here an extended description of the results from the corresponding figure in the main manuscript ( Figure 3B ). Briefly, we found a tendency for clonal expansion of the nTreg product derived clonotypes in the majority of the patients (Low dose: C5051, C5052, C5054, medium dose: C5058-R1B, and high dose: C5062, C5063-R and C5067), although some resisted this pattern (Low dose: C5053, medium dose: C5056-RIII and C5057-R1B, and high dose: C5059). Based on the sheer number of clonotypes, it is difficult to get an impression of the structure of the repertoire. The visualization in Figure 3B makes use of the division of clonotype frequencies into different groups (termed binning) and then to sum up the frequencies in each bin. The binning is essentially arbitrary, and here we used the bins 1, 0.1, 0.01, 0.001, and 0% (Please see the corresponding legend: TCR clone frequency). At some visits we observed hyper-expanded clonotypes, which can be related to as an artefact occurring due to the amplification process.. However, when we looked at the bins, in which we find less than 5 clonotypes, we see that the problem is relatively small, affecting only 5/47 samples (10%):

This section uses different mathematical expressions to describe the clonal diversity over time: either Renyi diversity profiles, or Shannon and Berger-Parker comparisons of diversity ( Figure S5C -H) (22-24).

The Renyi diversity profiles (22) combine different diversity measures to give a complete overview of the sample diversity. The x-axis indicates the weighing of clonotypes; at 0 all clonotypes are considered, and at "infinite" only the single most dominant clonotype is considered. Although somewhat complex, the interpretation is straightforward: 7 the sample that is always above all other samples is more diverse. If the lines of samples cross, their diversity cannot be distinguished. The diversity profiles here compare the different time points for each patient, first for all clonotypes ( Figure S5C ) and then for the top 500 clonotypes ( Figure S5D) . Overall, we did not find a particular pattern in diversity. No sample always had a different diversity compared to the others. This finding is not in direct contrast to the observation in Figure 3B , as we generally observe a decrease in diversity over time.

We next compared the diversity to the number of cells originally infused, with the most commonly used Shannon and Berger-Parker diversity (23, 24) . When comparing the Shannon diversity ( Figure S5E ) or the Berger-Parker index ( Figure S5F ) to the dose of cells infused at different time points, no association was indicated. We next compared rejection vs. nonrejection ( Figure S5G-H) . Four patients experienced signs of acute rejection or IgAN. To see if these events could be associated with diversity, we evaluated the Shannon and Berger-Parker diversity over time. There was no particular indication of association between diversity and rejection. We furthermore found no strong correlation between Treg frequency and sample diversity ( Figure S5J+K ). In most cases, the Treg frequency and Shannon diversity decrease over time, but in three cases (C5057-R, C5058-R, and C5059), the Treg frequency increased over time ( Figure S5K ).

Similarity indices measure the similarity of two populations considering not only the number of shared clonotypes but also take the clonotype count or percentage into account. One of the most popular indices is called Morisita-Horn similarity index (25). It is one of the first of the similarity indices, which can handle comparison of populations of unequal size (e.g. it is possible to compare a population of 100 clonotypes with a population of 20). The heatmaps of the similarity index are symmetric, there is the same number of columns and rows, and the upper triangle is identical to the lower triangle ( Figure S5L+M) . Numerically, the observed overlaps are small, but considering the potential repertoire being sampled, the chance of an overlap is very small. There is generally a larger overlap between the time points V04 and V06, than between any other time points. There are two exceptions, patient number C5053, with an overlap between V08 and V10, and patient 5067, with a stronger overlap between P and V04. The predominant overlap between V04 and V06 could be an indication of still settling homeostasis.

Comparison to product: To gain insight into the product-derived clonotypes, the total frequency of clonotypes found in the product and at later visits was evaluated ( Figure S5N) . A different question along the same line was how the product-derived clonotypes distribute across the visits ( Figure S5O ). For the low dose, there is a tendency to find more productderived clonotypes at the later visits, whereas for the medium and high-dose group the earlier visits seem to dominate regarding product-derived clonotypes. 

Figure S1: Trial Reporting and Assessment of Adverse Events and Efficacy in Patients.

(A) CONSORT Transparent Reporting of Trials for: The ONEnTreg13 Charité natural thymus regulatory T-cell (nTreg) therapy trial and The ONErgt11 Charité reference group trial; and (B) Assessment of adverse events and efficacy in patients: Different types of adverse events (AE) are labeled in the Venn diagram, with events within the yellow box being "related" to the investigational medicinal product, and "serious" adverse events in the red box, and "unexpected" adverse events in the blue box not yet being documented in the product information of the investigator brochure (IB). The primary clinical endpoint (biopsyconfirmed acute rejection, BCAR) requires histological confirmation from a for-cause tissue biopsy. Italicized diagnoses correspond to histological categories of renal allograft pathology defined by the Banff criteria (please see IB). There were no significant differences in the incidence of Severe Adverse Effects (SAEs). In summary we registered 14 SAEs in 10/11 patients of the ONEnTreg13 group. These SAEs were not categorized as directly related to the nTreg infusion (one mixed acute rejection was indirectly related to the weaning strategy in the nTreg group but could be resolved -weaning failure). In the rONErgt11-CHA reference group 12 SAE´s were noted in 9/9 patients. The figure is composed of n=11 individual panels giving an overview of all patients treated with nTregs in the ONEnTreg13 trial (C5051, C5052, C5053, C5054, C5056, C5057, C5058, C5059, C5062, C5063, and C5067. The panels are structured as follows:

At the top of each panel, detailed informative clinical parameters are given on the individual patient background in the context of kidney transplantation (donor sex and age, recipient sex and age, recipient body mass index (BMI, kg/m 2 ), time on dialysis (months), HLAmismatches (number), underlying disease, and any occurrence of complications of surgical, infectious immunological or other kind).

The center of each panel, shows the cell dose applied for each individual patient (either 0.5, 1.0, or 2.5-3.0 x 10 6 cells/kg) with a very brief summary of the clinical course (e.g. successful tapering of immunosuppression (IS) with stable creatinine) and a chart of the serum creatinine levels (mg/dL) over time (days after renal transplantation), together with the duration of IS (tacrolimus, mycophenolate-mofetil (MMF) and steroids) also indicating the tapering thereof.

The bottom of each panel, shows the results for the assessment of cellular and humoral allosensitization of the patient, with results of the IFN-gamma ELISpot assay (x spots/300,000  cells) being depicted on the left (IFN-gamma producing cells within donor-or patient-derived  PBMCs, with indication of patient visits V00, V03, and V09, and tested antigen: either  alloreactivity against donor, or antigen-reactivity against viral antigens pp65, IE-1, EBV, or SEB, see appreciations below) and the results of the panel-reactive antibody (PRA) screening being depicted to the right (positivity for HLA class I and II at patient visits V00, V03, V09, and V10, together with detailed results for specification indicated below, if positive), respectively.

Abbreviations Figure S3 : ACR, acute cellular rejection and AMR, antibody-mediated rejection with Banff-grade I-III; CNI, calcineurin-inhibitor; DSA, donor-specific antibodies; IgA-N, IgA nephropathy; IVIG, intra-venous immunoglobulin; MMF, mycophenolatemofetil; pp65 and IE-1, cytomegalovirus protein pp65 and IE-1 eliciting a CD8+ T-cell response; EBV, Epstein-Barr virus antigen; and SEB, staphylococcus enterotoxin B, a bacterial superantigen, and TNTC, to numerous to count (>max). (A) Flow chart of the natural regulatory T-cell (nTreg) manufacturing process, Briefly, the process is composed of three major steps (left panel): 1) Preparation of the starting population on day 0, 2) Cell expansion under several bead (re)-stimulations at day 1-22, and 3) Product filling and application on day 23, entailing magnetic bead-depletion, washing, filling and application. All process steps are accompanied by various process-controls (right panel), and (B) GMP-process approved and validated flow cytometry gating and analysis strategy with representative scatterplots / histograms applied for the quality control and functional characterization of nTreg products. In a first step, cellular "duplets" are excluded by gating for "singlets" within the forward scatter (FSC INT / FSC TOF) profile, followed by setting the lymphocyte-gate in the forward-sideward scatter (FSC / SSC) profile, followed by gating of "living" cells with side-ward scatter live-dead discrimination (SSC / LD), which is then followed by stepwise phenotypic T-cell subset analysis (center panel) with identification of CD3+/CD4+/CD25+/FoxP3+ regulatory T-cells of naïve or memory phenotype (CD45RA positive or negative, respectively), with concomitant functional characterization by detection of cytokine production (IL-2 and IFN-gamma) following maximal PMA/ionomycin stimulation, demonstrating the absence or very low expression of the respective Th1-related effector mediators (histograms to the right). The following sequence of panels in Figure S5A -O gives further bioinformatics evaluation for the initial TCR-sequencing data quality control and the clonal diversity and overlap over time. Depending on the underlying analysis, data are grouped either according to patient visits (V04, V06, V08, V10, and "GMP" for the GMP-expanded nTreg product prior to infusion), and/or infused cell dose (low, medium and high) and/or patient rejection status ("R", all four patients with potential rejection event are marked: C5056R, C5057R, C5058R, and C5063R; C5063 only had IgAN, a rejection like pathology). In principle, there are three major aspects/questions answered in the following figures:

12 1) Initial Data Quality Control: (A+B) Number of clonotypes and of the accepted reads are sorted according to time points to check any negative bias from unacceptably low reads. Renyi diversity profiles for each patient and time point  depicted either for all clonotypes or for the top 500 clonotypes, with each line depicting one  time point; (E-F) Shannon and Berger-Parker diversity for each infusion group (low, medium, and high cell dose) according to patient visits; (G+H) Shannon and Berger-Parker diversity over time of visits for each patient with distinction of rejection and non-rejection; and (J+K) Correlation of Shannon diversity to Treg frequency and association between Treg frequency and Shannon diversity). see  Table S5 . The figure is composed of three different parts:

Part 1) FACS data for the following immune subset parameters: #01) Blood differential counts, #02) Monocytes, #03) Dendritic cells, ##04) TCR-γ/δ T-cells, #05) Natural killer (NK) and T cells, #06-11) CD4 and CD8 T-cell subsets, activation, memory, CD4 regulatory T cells (Tregs), and Treg to Tconv ratios, and #12) B cells, all expressed either as absolute counts (cells/nl, left column) or cellular frequencies (% of parent, right column), for two different general comparisons: a) nTreg-treated vs. reference group (shown at the left), and b) nTreg dose-response analysis (shown to the right). Part 3: qPCR data of whole blood samples: Multiple gene expression markers (n=20 targets) were studied on mRNA transcript level with qPCR, as stratified in the study below, again for the same two general comparisons: a) nTreg-treated vs. reference group shown to the left, and b) nTreg dose-response analysis shown to the right. The establishment of the tolerance/rejection signature gene expression panel is described in the following reference:

13 

The primary objective of the ONEnTreg13 phase I/IIa trial was to assess safety and feasibility of the first-in-human application of our in-house developed autologous CD4+CD25+FoxP3+ nTreg product in living-donor KTx patients (n=11). Results were compared to the reference patients of our centre in the ONErgt11-CHA trial (n=9), conducted prior to the ONEnTreg13, to establish safety margins and biomarker panels for the study.

Incidence of biopsy-confirmed acute rejection within 60 weeks of organ transplantation (histological grading according to the Banff criteria).

To assess the safety and feasibility of intravenous infusion of ex vivo-selected and ex vivoexpanded autologous natural Tregs in patients with solid organ transplantation.

• adverse infusion-related effects • infections, response to anti-rejection therapy • graft function/failure Secondary Objective

To evaluate the effect of nTregs on solid organ graft function (e.g. kidney graft) during tapering of immunosuppression as compared to the reference group receiving conventional triple maintenance immunosuppression.

• Surrogate markers of transplant function and surrogate immunologic markers related to general immune function. • Time to first acute rejection episode • Severity of acute rejection episodes based on response to treatment and histological scoring • Return to transplant waiting list or re-transplantation following graft-loss due to rejection (acute or chronic) • Incidence of adverse drug reactions • Incidence of post-transplant dialysis • Immunosuppressive burden at final visit 

• Chronic renal insufficiency necessitating kidney transplantation and approved to receive a primary kidney allograft from a living donor.

• Age 18 years or older and written informed consent.

• Patient previously received a tissue or organ transplant other than the kidney graft.

• Known contraindication to the protocol-specified treatment/medications.

• Genetically identical to the prospective organ donor at the HLA gene loci *.

• PRA grade >40 % within 6 months prior to enrolment.

• Previous treatment with any desensitization procedure (with or without IVIG)

• Concomitant malignancy or history or malignancy within 5 years prior to planned study entry (excluding successfully treated non-metastatic basal/squamous cell carcinoma of the skin).

• Evidence of significant local or systemic infection.

• HIV-positive, EBV-negative or suffering from chronic viral hepatitis. • For nTreg preparation ca. 50 ml patient blood is collected to Lithium-Heparin blood collection tubes by venipuncture. •

• Depletion of CD8 + T cells and enrichment of CD25 + cells from the CD8-depleted cell fraction.

.

• After isolation, the cells are cultivated at 37°C in a 5% CO2 atmosphere. • The cell expansion phase is initiated and maintained by (re) stimulation with anti-CD3/anti-CD28 antibody coated GMPgrade Treg expansion beads. • Cell splitting and/or medium exchange is realized on the basis of visual culture inspection and cell counting. • The release-criteria-testing is carried out on the aliquots taken during manufacturing.

In-process

• On day 23, when the target cell number is reached, cells are washed and resuspended in PBS. Beads are removed by LD-Columns.

end product

• After bead-removal, cells are washed in saline. One batch will result in 50 ml cell-solution filled in a perfusor syringe. • The amount of cells depends on the body weight of the patient and the dose, which is defined in the clinical protocol. end product 16 16 To facilitate data-interpretation, all statistical subset definitions and statistical testing methods (described in more detail in the statistics sections of the main manuscript and published in the reference below) and the obtained P-values are summarized in the following overview tables: Table S6A : Nonparametric analysis of time-course: clinical parameters Table S6B : Overview of immune cell populations Table S6C : Nonparametric analysis of time-course: absolute leukocyte subset counts Table S6D : Nonparametric analysis of time-course: leukocyte subset frequencies 

Leucocytes Table S7 : Asterisks and numbers denote: * optional assessment, and ** to be performed 6 hours after cell infusion; 1) Only for female patients; 2) Heart rate, blood pressure, and temperature measurements, with heart rate and blood pressure being monitored after 5 min of rest and according to local trial center routines; 3) Patient mass (kg) and height (m) should be measured to calculate the BMI: formula = mass / (height) 2 ; 4) ICC (International Intensive Care) monitoring of the patient for nTreg cell infusion includes ECG, blood pressure, respiratory rate, SaO 2 and temperature; 5) Red blood cells, hemoglobin, hematocrit, mean cell volume, white blood cells (with differential count) and platelets; 6) Na + , K + , Table S5 • Part 1: FACS Immune Cell Subsets for the Following Parameters: 

Mul?ple hypothesis-driven gene expression markers (n=20 targets) were studied on mRNA transcript level with qPCR, as stra?fied in the study below, again for the same two general comparisons: a) nTreg-treated vs. reference group shown to the leO, and b) nTreg dose-response analysis shown to the right Figure S6 : Part 1 FACS#05: TCR-γ/δ T-cells 

generated using the standardized ONE-Study protocol

Cross-validation of IFN-gamma Elispot assay for measuring alloreactive memory/effector T cell responses in renal transplant recipients

Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing

Applications of next-generation sequencing to blood and marrow transplantation

TCR repertoire analysis by next generation sequencing allows complex differential diagnosis of T cell-related pathology