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Correspondence: Hendrik Christian Albrecht, Department of General, Visceral, Thoracic and Vascular Surgery, University Hospital Ruppin-Brandenburg, Neuruppin, Germany.
Department of General, Visceral, Thoracic and Vascular Surgery, University Hospital Ruppin-Brandenburg, Neuruppin, GermanyFaculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany
Department of General, Visceral, Thoracic and Vascular Surgery, University Hospital Ruppin-Brandenburg, Neuruppin, GermanyFaculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany
Center of Internal Medicine II, University Hospital Brandenburg, Brandenburg, GermanyFaculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany
Institute of Pathology and Cytology, University Hospital Ruppin-Brandenburg, Neuruppin, GermanyFaculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany
Medical Department, Divisions of Gastroenterology, Hepatology, Oncology, Hematology, Endocrinology, University Hospital Ruppin-Brandenburg, Neuruppin, GermanyFaculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany
Department of General, Visceral, Thoracic and Vascular Surgery, University Hospital Ruppin-Brandenburg, Neuruppin, GermanyFaculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany
Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 109 cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3+/CD8+ effector memory phenotype (CD45RO+/CCR7−). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay)
Colorectal carcinoma (CRC) is one of the most common malignant tumors of the digestive tract and a relevant cause of cancer-related deaths. It is the third most-common tumor disease in both sexes worldwide and the second-leading cause of death among all cancers [
]. Over the decades, classical treatment of these tumors, i.e., surgical excision, standard chemotherapy and radiation, has been refined, resulting in significantly prolonged survival, especially for tumors detected in early stages of disease. Despite screening programs and preventive strategies, approximately 25% of patients still present with late tumor stages. Advances in surgery and expansion of systemic treatment options achieve only low improvement of median survival in patients with metastatic CRC. In recent years, mortality decreased slightly in older patients (>50 years) but remained stable in younger patients (20–49 years) [
There is a need for effective therapeutic strategies, especially in chemorefractory tumors. CRCs with high mutational burden respond to immunotherapy. Immune checkpoint inhibitors (programmed death-1–specific antibodies as pembrolizumab or nivolumab) gain durable responses in mismatch repair-deficient-microsatellite instability-high (dMMR-MSI-H) CRC [
Recently, adoptive cell transfer (ACT) using tumor-infiltrating T lymphocytes (TILs), chimeric antigen receptor insertion, or T-cell receptor modification has emerged as a promising approach for tumor therapy. ACT can be used to harness the host T-cell immune response for immunotherapy in which ex vivo mass-expanded activated TILs from resected individual tumors are reinfused to the patient. ACT using TILs achieved durable complete remission in 20%–25% of patients with metastatic melanoma [
Previous studies showed that interactions among the immune microenvironment and the mediated immune response are related to a favorable prognosis in CRC [
]. A high degree of tumor-infiltrating lymphocytes (TILs) in specimens is associated with decreased tumor invasiveness, less nodal involvement and improvement in survival [
Despite the enormous disease burden and large number of premature deaths caused by CRC, and despite the good rationale for an immunotherapeutic approach, this entity has been only a minor object in cell therapy research [
]. Moreover, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations.
The aim of this study was to investigate the feasibility of isolating TILs from CRC tissue samples and mass expanding of TILs for ACT in a controlled two-phase process in a perfusion bioreactor. The expanded TILs were characterized according to phenotype, and activity was determined based on cytotoxicity to different stimulations.
Methods
Tumor samples
Tumor tissue samples were obtained from patients with primary CRC undergoing surgical resection without preoperative chemotherapy, radiotherapy or immunotherapy at the Department of General, Visceral, Thoracic, and Vascular Surgery, University Hospital Ruppin-Brandenburg, Brandenburg Medical School, as part of a study to analyze essential fatty acids/lipid mediators and their function in solid tumors (Ethics Committee of the Brandenburg Medical School No. Z-03-20170508). All patients gave their written informed consent to participate in the study, and included were 12 primary colon tumors obtained by surgical resection with curative intent. Additional clinical data of patients are listed in Table 1. After surgical resection, tumors were localized in the specimen and opened by an incision from the luminal or extraluminal side. The tumor center, invasive margin, and peritumoral environment were identified, and tissue samples were collected from the invasive margin area.
Pathological characterization and transportation to in vitro processing
TILs were quantified in the obtained tissue samples before expansion and their phenotype was characterized. For this process, paraffin-embedded tissue samples fixed in formalin were used and hematoxylin-eosin (H&E)-stained slides were prepared.
The standardized method for the assessment of TILs in solid tumors on H&E sections was applied according to the guidelines of the International Immuno-Oncology Biomarker Working Group [
Assessing tumor-infiltrating lymphocytes in solid tumors: a practical review for pathologists and proposal for a standardized method from the international immunooncology biomarkers working group: part 1: assessing the host immune response, TILs in invasive breast carcinoma and ductal carcinoma in situ, metastatic tumor deposits and areas for further research.
]. Semiquantitative H&E evaluation of the TILs was performed at 200–400× magnification. In addition, tissue samples were examined by immunohistochemistry for infiltration with CD4- and CD8-positive T cells (supplementary Figure 1).
The following antibodies were used for immunostaining: monoclonal mouse anti-human CD4 (Agilent Technologies [Santa Clara, CA, USA], 4B12, ready-to-use) and monoclonal mouse anti-human CD8 (Agilent Technologies, C8/144B, ready-to-use).
The immuno-stained slides were analyzed semi-quantitatively by a pathologist, who assessed the ratio between the number of positively stained cells and the total number of cells in five high power fields. Areas with significant necrosis, hemorrhage, as well as histologic artifacts, were excluded from analysis.
Tumor tissue samples from each donor were transported to the laboratory under antibiotic-antifungal conditions (CellGenix GMP DC Medium [Sartorius CellGenix, Freiburg, Germany], 10% human serum [PAN Biotech, Bavaria, Germany], 100 units penicillin, 0.10 mg streptomycin and 0.25 μg amphotericin B per mL [Sigma–Aldrich, St. Louis, MO, USA]).
As a result of contamination events during primary cultivation and the loss of two tumor samples, the transportation medium was stowed with double-concentrated antibiotic-antimycotic reagent (200 units penicillin, 0.20 mg streptomycin and 0.50 μg amphotericin B per milliliter).
Activation and expansion of TIL in perfusion bioreactor mode
Preparation of tumor tissue, cultivation and ex vivo expansion of tumor infiltrating lymphocytes was done in a proprietary cultivation process developed by Zellwerk GmbH [
Hoffmeister, H. (2019). Meander bioreactor and method for the isolation and proliferation of cells from parts from tumors, metastases and other tissues. Germany patent application.
Hoffmeister, H. (2020). Meandering perfusion bioreactor for the differentiation, activation, stimulation and separation of cells. Germany patent application.
]. Tumor tissue material was sliced into 2 × 2 × 2-mm pieces and filled into 15 mL of complete cultivation medium (CCM), composed of CellGenix GMP DC Medium (Sartorius CellGenix), 10% human serum (PAN Biotech), 1% antibiotic antimycotic solution (100 units penicillin, 0.10 mg streptomycin and 0.25 μg amphotericin B per milliliter) (Sigma-Aldrich) and 1000 IU/mL human IL-2 (Proleukin; Clinigen, Burton upon Trent, UK). After adding 10 ng per milliliter of human IL-12 (premium grade; Miltenyi Biotec, Bergisch Gladbach, Germany), the suspension was transferred via port directly into a perfusion bioreactor. Outgrowth, activation and expansion of TIL were carried out in a single use, GMP-compliant and completely closed perfusion bioreactor 30MM. The bioreactor runs were operated on a platform of GMP breeder, Control Unit (both from Zellwerk GmbH, Oberkrämer, Germany) and associated software [
]. This platform allows measurement and control of medium temperature, pH and pO2 (set point for temperature: 36.5°C; for pH: 7.2–7.3 [regulated by CO2 influx]; for pO2: 25% [using a gas mixture of 25% O2 and 75% N2]).
The first cultivation period within a bioreactor run comprised 6–8 days. Inoculation was performed with different numbers of tissue pieces in CCM with 10 ng per milliliter human IL-12 under static aeration conditions (5% CO2, 25% O2, 70% N2). In the first two experiments, we inoculated 50 tissue pieces, each in a total volume of 400 mm3. Later, total tissue volume was reduced to 240 mm3 (30 pieces, four donors) and subsequently, from each of 5 donors 10 pieces (80 mm3) were inoculated, leading to the best yield. At days 3–5 within the first cultivation period, outgrowth and expansion of TIL is initiated by a short activation burst: A cocktail containing 30 ng per milliliter anti-human CD3 antibody (clone OKT3; Miltenyi Biotec) and a number of 2 million allogenic human feeder cells per tumor piece (irradiated with 55 Gy), is infused once into a bioreactor. During the next 3–5 days, an accelerated outgrowth of TILs is observed.
The second cultivation period begins with starting medium circulation in the bioreactor vessel as well as a continuous influx of fresh CCM. The culture process is from this time automatically controlled by an algorithm based on continuous measurements of pH, pO2 and temperature in the circulating medium and provides upgrowing cells with fresh medium.
As CCM is only supplemented with interleukin (IL)-2, the activating substance IL-12 is washed out after approximately 1 day of fresh CCM supply. Medium circulation and needed fresh medium influx over the whole expansion phase of TIL until harvesting is steered automatically, guaranteeing homogenous nutrient supply. To achieve a rolling movement, changing the touching areas between the sedimented TIL, the bioreactor vessel needs a short gently shaking every 5–7 days.
Over the whole culturing time, supernatant samples were regularly taken and concentrations of glucose and lactate were routinely measured using a biochemical analyzer (YSI 2700 Select, YSI).
When the lactate concentration reached a target value of 1 g/L, influx of fresh medium (perfusion rate) was adjusted manually to maintain this value. Cells were expanded until an average final daily lactate production of 80–120 mg per day was reached. The cell number reached was approximately 1.5 to 2.5 × 109 within a period of 3–4 weeks. The bioreactor was shaken to suspend the cells before taking samples for cell counting. Cells were harvested, contemporaneous separated from tissue material by transferring them by gravity over the mesh port, cryopreserved and stored in liquid nitrogen until used for the second expansion phase.
For the second expansion phase, cells were thawed, washed and mixed with 20 mL of complete cultivation medium (as mentioned previously) and filled directly into the bioreactor. The bioreactor was inoculated with approximately 300 × 106 cells. After 3 hours, when cells had settled down, medium circulation and aeration were enabled. During this expansion, the observed process parameters were comparable to those of the first expansion phase. The tumor volume or inoculated cells used for the first or second expansion phase, the duration of cultivation and the yield of harvested cells are listed in Table 2.
Table 2Cultivation parameters: Starting material, cell yield, duration of cultivation, and metabolites in bioreactor in first and second expansion phases.
The phenotype of expanded cells of each bioreactor cultivation was analyzed by using a BD Accuri C6 Plus (BD Biosciences, Franklin Lakes, NJ, USA) or MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). Characterization of different lineage markers and other expressed surface markers was performed directly after bioreactor harvesting as well as after short-term cultivation of cryopreserved cells.
For analysis, cells were washed, followed by staining with different sets of antibodies. Anti-CD3 PerCP-Cy5.5 (clone: UCHT1; BioLegend, San Diego, CA, USA), anti-CD4 FITC (clone: RPA-T4; BioLegend), anti-CD8 FITC or PE (clone: SK1; BioLegend), anti-CD28 APC (clone: CD28.2; BioLegend); anti-CCR7 APC (clone: G043H7; BioLegend), anti-CD25 APC (clone: M-A251; BD Biosciences); anti-CD27 PE (clone: M-T271; BD Biosciences); anti-CD45RO PE (clone: UCHL1; BD Biosciences); anti-CD56 FITC (clone: B159; BD Biosciences); anti-CD127 PE (clone: HIL-7R-M21; BD Biosciences); anti-CD183 PE (clone: 1C6/CXCR3; BD Biosciences); anti-CD196 APC (clone: 11A9; BD Biosciences) (panel 1) or Anti-CD3 PE-Vio615 (clone: REA613), anti-CD4 APC-Vio770 (clone: REA623), anti-CD8 VioGreen (clone: REA734), anti-CD25 PE (clone: REA945); anti-CD27 PE (REA499); anti-CD28 APC (REA612); anti-CD45RO FITC (clone: REA611); anti-CD56 APC (clone: REA196); anti-CD127 APC (clone: REA614); anti-CD183 APC (REA232); anti-CD196 PE-Vio770 (REA190); anti-CCR7 PE-Vio770 (clone: REA546) (panel 2, all Miltenyi Biotec) were used as lineage markers for T cells (Th, Tc, Treg) and NK cells, and combined with either T cell subtype markers for naïve, central memory (TCM), effector or effector memory T cells (Teff, TEM) and for T helper cells (Th1, Th2, Th17). Cells were stained for 30 min at room temperature, washed and finally re-suspended in fresh staining buffer.
Cell culture
Tumor-infiltrating lymphocytes
Before any analytical measurements, cryopreserved TILs were re-vitalized in short-term culture. The cells were thawed, followed by washing (350g, 5 min) with pre-warmed cultivation medium. Cells were then resuspended in CCM, transferred into flasks and cultivated for 48 h at 37°C, 25% O2, 5.5% CO2. Afterwards, cells were collected, centrifuged (350g, 5 min) and resuspended in TIL-CCM.
CRC cell lines
For co-cultivation of TIL and CRC cell lines, cryopreserved aliquots of each cell line (COLO678 [ACC194], HCT116 [ACC581], JVE127 [ACC813]; DSMZ) were thawed and then mixed with prewarmed cultivation medium (CRC-M; RPMI-1640 [Gibco/Thermo Fisher Scientific, Waltham, MA, USA], 10% fetal bovine serum [Sigma-Aldrich], 1% antibiotic antimycotic solution (100 units penicillin, 0.10 mg streptomycin and 0.25 μg amphotericin B per milliliter) [Sigma Aldrich]). Cells were then resuspended in CRC-M, transferred to flasks and cultured at 37°C and 5.5% CO2.
Functional assays
Qualitative interferon (IFN)γ staining assay
The potential of TILs to express IFNγ was analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). To assess the intracellular IFNγ expression of TILs, cells were cultivated as described previously. After short-term cultivation, cells were resuspended in complete supplemented medium (included 10% human serum, 1% antibiotic antimycotic solution, IL-2) in a concentration of 2 million cells per milliliter. Then, 50 µL of this suspension were transferred into 96-well round-bottom plates. Cells were stimulated with anti-CD3 antibody (clone: OKT3, Miltenyi Biotec) at a final concentration of 30 ng per milliliter. Phorbol-12-myristat-13-acetat (PMA) was used as an alternative stimulation reagent and as a positive control (final conc. 2.5 µg/mL). After 1 h of incubation at 37°C, 25% O2, 5.5% CO2, 50 µL of GolgiStop (1:150, BD Biosciences) and GolgiPlug (1:100, BD Biosciences), diluted in complete supplemented medium (included 10% human serum, 1% antibiotic antimycotic solution, IL-2, anti-CD3 Ab), were added. After further 5 h of incubation, cells were collected, centrifuged and incubated together with TruStain FcX (BioLegend) for 10 min at room temperature. Afterwards, surface markers were stained with anti-CD3 PerCP-Cy5.5, anti-CD4 FITC and anti-CD8 PE antibodies for 30 min at room temperature (Ab details described previously: panel 1). The fixation/permeabilization (BD Cytofix/Cytoperm fixation/permeabilization kit; BD Biosciences) overnight at 4°C was performed after washing with staining buffer (350g, 5min). At the next day, cells were washed with perm/wash solution and stained with anti-IFNγ APC (clone: 4S.B3, BioLegend) antibody for 30 min at room temperature. After final washing, cells were resuspended in staining buffer and stored at 4°C until measurement.
Quantitative cytokine assay
The concentrations of secreted cytokines were analyzed by using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). To assess the potential of TILs to secret different cytokines, cells were cultivated as described previously. After short-term cultivation, cells were re-suspended in complete supplemented medium (included 10% human serum, 1% antibiotic antimycotic solution, IL-2) in a concentration of 1 million cells per milliliter. Then, 200 µL of this suspension were transferred into 96-well round-bottom plates. Cells were stimulated with anti-CD3 Ab at a final concentration of 30 ng/mL (clone: OKT3, Miltenyi Biotec). Further, after 6 h of incubation, supernatants were collected, centrifuged (10 000g, 1 min) and the cell-free supernatants were frozen at –20°C.
To measure different cytokines in parallel, Miltenyi Biotec MACSPlex cytotoxic assays were used. In this assay, we combined the following products, MACSPlex mix cytotoxic basic kit, MACSPlex mix cytotoxic standard and MACSPlex mix cytotoxic reagents kits for IFNγ and tumor necrosis factor (TNF)α.
At the day of measurement, supernatants were thawed, centrifuged (10 000g, 10 min) and prepared as described by the manufacturer. To analyze and quantify, amounts of cytokines were measured by MACSQuant Analyzer Express Mode for MACSQuant Instrument.
Degranulation and intracellular cytokine assay
Degranulation and intracellular cytokine expression were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). To investigate the degranulation and intracellular cytokine expression (IFNγ, TNFα) of TILs induced by tumor-cell-specific stimuli, three CRC-cell lines were co-cultivated with TILs. Two to three days before co-cultivation, CRC cell lines were resuspended in CRC-M (RPMI-1640, 10% fetal bovine, 1% antibiotic antimycotic solution) at a concentration of half a million cells per milliliter. Then, 100 µL of this suspension was transferred into 96-well flat-bottom plate and cultivated at 37°C, 5.5% CO2. On the day of co-cultivation, short-term TILs (as described previously) were resuspended in complete supplemented medium (containing 10% human serum, 1% antibiotic antimycotic solution and IL-2) at a concentration of 10 million cells per milliliter. Then, 50 µL of this suspension was transferred into 96-well flat-bottom plates containing CRC-cell lines (after removal of CRC medium). CD107a-Ab (anti-CD107a APC [REA792]; Miltenyi Biotec) and/or PMA (duplicates for each CRC-cell line as stimulation control; final concentration 2.5 µg/mL) were transferred into TIL-CRC-cocultures. After 1 h of incubation at 37°C, 25% O2, 5.5% CO2, 20 µL of GolgiStop (1:132, BD Biosciences) and GolgiPlug (1:99, BD Biosciences), diluted in complete supplemented medium, were added. After further 5 h of incubation, cells were collected, centrifuged and washed with staining buffer (autoMACS Rinsing solution incl. BSA; Miltenyi Biotec). Cells were then fixed and stained as described by the manufacturer (extracellular: anti-CD3 PE-Vio770 [REA613], anti-CD4 APC-Vio770 [REA623], anti-CD8 VioGreen [REA734]; intracellular: anti-IFNγ FITC [REA600] and anti-TNFα PE [REA656]; all antibodies from Miltenyi Biotec). After the final wash, cells were resuspended in staining buffer and immediately measured. Each TIL-CRC co-culture was analyzed in triplicate.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA). Statistical differences between unstimulated and stimulated samples were determined using a nonparametric one-way analysis of variance rank test (Friedmann) with Dunn multiple comparisons test.
Results
Pathological screening
Pathological screening of the obtained tumor samples showed a mean TIL infiltration rate of 34% (5%–60%) using H&E staining. Immunostaining revealed a mean rate of 62% (20%–95%) CD4+ T cells and of 30% (5%–50%) CD8+ T cells (Table 3, supplementary Figure 1).
Table 3Percentage frequency of TIL subpopulations: Tissue samples were pathological screened by H&E and immunohistochemical staining.
Subtypes
Donor
Surface marker
TIL-52.1
TIL-54.1
TIL-55.1
TIL-57.1
TIL-63.1b
TIL-56.1
TIL-59.1
TIL-61.1
TIL-63.1a
TIL-66.1
TIL-68.1
Pathology
Infiltrating T cells
10
50
10
50
50
30
60
12
50
60
50
CD3+
Th
95
80
20
50
50
80
50
70
50
50
70
CD4+
Tc
5
40
40
50
50
20
50
20
50
30
20
CD8+
First phase
NK
N/A
N/A
1.5
0.3
3.0
0.0
0.1
0.2
0.7
0.3
0.1
CD3− CD56+
Th
9.3
0.6
55.2
26.0
39.8
24.5
18.6
55.3
29.8
CD3+ CD4+
Tc
32.5
84.5
45.8
72.9
65.7
77.9
72.0
57.4
74.2
CD3+ CD8+
TDP
0.2
0.0
1.4
0.2
0.2
17.3
0.5
0.7
0.8
CD3+ CD4+ CD8+
CD4_TEM
7.3
0.8
48.2
21.1
30.6
25.1
17.5
48.3
28.1
CD3+ CD4+ CD45RO+ CCR7−
CD4_Teff
0.2
0.2
0.0
0.3
0.0
0.1
0.0
0.0
0.2
CD3+ CD4+ CD45RO− CCR7−
CD4_TCM
2.3
0.1
5.6
0.7
0.8
0.6
3.0
4.1
0.5
CD3+ CD4+ CD45RO+ CCR7+
CD4_Treg
1.7
0.6
1.1
1.3
1.2
0.8
1.6
2.3
0.2
CD3+ CD4+ CD25+ CD127-
CD4_naïve
0.1
0.2
0.1
0.0
1.0
0.0
0.0
0.1
0.0
CD3+ CD4+ CD45RO- CCR7+
Th1
7.8
0.5
25.9
17.0
29.3
17.6
12.0
23.0
19.8
CD3+ CD4+ CD183+ CD196-
Th2
1.0
0.1
2.5
5.8
4.9
4.6
5.3
18.6
2.4
CD3+ CD4+ CD183- CD196-
Th17
0.0
0.1
4.9
0.3
0.7
1.6
1.1
6.2
0.8
CD3+ CD4+ CD183- CD196+
CD8+ TEM
25.0
74.3
42.4
71.0
63.9
67.3
60.3
55.7
72.6
CD3+ CD8+ CD45RO+ CCR7−
CD8+ Teff
6.3
1.4
0.0
0.4
0.5
8.3
0.2
0.6
0.9
CD3+ CD8+ CD45RO− CCR7−
CD8+ TCM
6.6
4.7
4.4
1.7
1.9
1.5
11.3
0.8
0.5
CD3+ CD8+ CD45RO+ CCR7+
CD8+ naïve
1.9
2.5
0.0
0.0
0.5
0.2
0.2
0.1
0.0
CD3+ CD8+ CD45RO- CCR7+
CD27+ Tc
1.7
0.2
20.0
2.8
11.4
5.4
14.4
19.2
33.5
CD3+ CD8+ CD27+
CD28+ Tc
8.5
5.6
27.2
10.6
49.2
16.5
41.8
37.5
43.6
CD3+ CD8+ CD28+
CD27+CD28+ TDP
0.6
0.0
13.8
0.6
8.9
2.0
10.7
20.8
28.7
CD3+ CD8+ CD27+ CD28+
Second phase
NK
N/A
N/A
N/A
N/A
0.1
0.1
1.1
0.0
0.2
CD3− CD56+
Th
1.7
12.6
3.3
40.5
4.0
CD3+ CD4+
Tc
95.1
93.4
92.1
55.6
88.8
CD3+ CD8+
TDP
0.1
2.3
0.2
0.8
1.0
CD3+ CD4+ CD8+
CD4_TEM
1.6
18.9
3.2
39.8
3.4
CD3+ CD4+ CD45RO+ CCR7−
CD4_Teff
0.0
2.5
0.0
0.0
0.5
CD3+ CD4+ CD45RO− CCR7−
CD4_TCM
0.1
0.6
0.3
0.6
0.1
CD3+ CD4+ CD45RO+ CCR7+
CD4_Treg
0.2
0.1
0.0
6.8
0.1
CD3+ CD4+ CD25+ CD127-
CD4_naïve
0.0
0.1
0.0
0.0
0.0
CD3+ CD4+ CD45RO- CCR7+
Th1
1.2
10.2
2.4
24.9
2.5
CD3+ CD4+ CD183+ CD196-
Th2
0.2
0.4
0.3
8.0
0.4
CD3+ CD4+ CD183- CD196-
Th17
0.1
0.0
0.0
4.5
0.3
CD3+ CD4+ CD183- CD196+
CD8+ TEM
94.4
36.5
85.7
53.6
80.4
CD3+ CD8+ CD45RO+ CCR7−
CD8+ Teff
0.2
19.7
2.4
1.3
6.1
CD3+ CD8+ CD45RO− CCR7−
CD8+ TCM
0.5
0.3
1.6
0.8
0.3
CD3+ CD8+ CD45RO+ CCR7+
CD8+ naïve
0.0
0.1
0.3
0.0
0.0
CD3+ CD8+ CD45RO- CCR7+
CD27+ Tc
12.9
2.3
4.4
22.2
11.3
CD3+ CD8+ CD27+
CD28+ Tc
95.4
0.8
2.5
54.6
88.1
CD3+ CD8+ CD28+
CD27+CD28+ TDP
12.9
0.3
0.6
22.2
11.3
CD3+ CD8+ CD27+ CD28+
Phenotypical characterization of cells from both expansion phases analyzed by flow cytometry.
The expansion of TILs in the proprietary ZRP bioreactor was designed as a two-phase process: an initial first expansion of TILs after insertion of multiple tumor pieces with a defined size all originating from a single primary tumor, followed by a second massive expansion of TILs after removal of all initially used tumor pieces. To define the appropriate total tumor volume as well as the defined size and number of tumor samples needed for the first expansion phase, the ZRP bioreactor was inoculated as follows. We tested the TIL expansion using two tumor tissues with a high total volume of 400 mm3 and cut equally into 50 pieces with an approximate volume of 8 mm3 each. Then, four tissues with an intermediate total volume of 240 mm3 and finally five tissues with a low total volume of 80 mm3 were tested. Taken together, 11 runs were performed using three different defined tumor volumes.
Total tumor volumes of 400 mm3 or 240 mm3 yielded only a low-to-moderate number of TILs after the first expansion phase, i.e., 10 million to 1.4 billion cells in six runs (Table 2/Figure 1A). Only one run using a specimen with 240 mm3 total tumor volume was able to produce more than 1 billion TILs, a defined intermediate goal of the first expansion and prerequisite for the second expansion phase. The 80 mm3 total tumor volume approach yielded an average of 1.752 (1.272–2.175) billion TILs in five runs. Notably, each run with 80 mm3 reached the prerequisite of 1 billion cells.
Fig. 1Cell suspension characterization expanded by 80 mm3 total tumor volume: Cell yield (A) and frequency of CD3-gated TIL subsets (CD4+, CD8+, CD4+ CD8+) after the first (B) and second (C) expansion phases.
A starting inoculation volume of 0.25–0.34 billion cells for the second expansion phase, yielded an average of 2.366 (1.125–3.100) billion TILs, in which four of five runs achieved more than 2 billion cells. The average cultivation time for expansion phase 1 was 25 days (17–32 days) and 20 days (17–23 days) for phase 2, respectively.
TIL phenotyping
All harvested cells were analyzed using flow cytometry (Table 3/Figure 1B,C). The first expansion phase yielded an average of 28.8 % (0.6%–55.3 %) CD4+ and 64.6 % (32.5%–84.5 %) CD8+ T cells in all 11 runs regardless of the total inoculated tumor volume. Of note, the proportion of CD8+ T cells increased on average to more than 85% during the second expansion phase in all five runs initially started with 80 mm3 total tumor volume. The proportion of CD4+ T cells levelled out at 12.4 % (1.7%–40.5 %).
Furthermore, we analyzed the effector (TE) and effector memory (TEM) phenotype of all harvested T cells (Figure 2). Regarding effector memory T cells, after the first phase 25.2% (0.8%–48.3%) CCR7− CD45RO+ CD4+ TEM cells and 59.2% (25.0%–74.3%) CCR7− CD45RO+ CD8+ TEM cells were reached. The second phase resulted in 12.0% (1.6%–39.8%) CCR7− CD45RO+ CD4+ TEM cells and 72.6% (36.5%–94.4%) CCR7− CD45RO+ CD8+ TEM cells. For effector T cells, the first phase yielded 0.1% (0.0% 0.3%) CCR7− CD45RO− CD4+ T cells and 2.1% (0.0%–8.3%) CCR7− CD45RO− CD8+ T cells. The second phase resulted in 0.6% (0.0% 2.5%) CCR7− CD45RO− CD4+ T cells and 5.9% (0.2%–19.7%) CCR7− CD45RO− CD8+ T cells (Figure 2).
Fig. 2Frequency of effector T cells (CD45RO− CCR7−) and effector memory T cells (CD45RO+ CCR7−) after the first (A) and second (B) expansion phases. A single dot represents percentage value of individual 80 mm3 tumor bioreactor expansion.
The effector function of harvested T cells was assessed by analyzing degranulation (CD107a-expressing) and/or cytokine expression (IFNγ as well as TNFα) after short-time stimulation with CRC-unspecific reagents like anti-CD3 antibody or PMA, and by co-culturing with CRC cell lines (JVE127, COLO678, HCT116) (Figure 3, supplementary Table 1, supplementary Figure 2, supplementary Figure 3).
Fig. 3Frequency of IFNγ expressing CD4+, CD8+ and CD4+CD8+ double-positive T cells: The expression of IFNγ was measured after TIL stimulation with anti-CD3 Ab (A) and PMA (B) in the first and second expansion phases. Single dots represent mean values of individual stimulation experiments (n = 5; mean ± standard error of the mean). Statistical differences between unstimulated and stimulated samples were determined using a nonparametric one-way analysis of variance rank test (Friedmann) with Dunn multiple comparisons test. **P = <0.01; ****P = <0.0001. The individual values of each tumor can be found in supplementary Table 1.
T-cell receptor complex/CD3-specific stimulation of T cells from the first expansion phase led to 0.8% (0.4%–1.3%) IFNγ + CD4+ T cells and 3.1% (2.3%–4.7%) IFNγ + CD8+ T cells. Moving to the second phase, stimulation resulted in 1.2% (0.4%–1.9%) IFNγ + CD4+ T cells and 4.2% (1.0%–6.6 %) IFNγ + CD8+ T cells. PMA stimulation of T cells from the first expansion phase led to 12.0% (7.1%–21.0%) IFNγ + CD4+ T cells and 50.6% (34.7%–66.0%) IFNγ + CD8+ T cells. Moving to the second phase, stimulation resulted in 19.0% (2.0%–44.7%) IFNγ + CD4+ T cells and 53.4% (22.7%–77.1%) IFNγ + CD8+ T cells (Figure 3, supplementary Table 1).
Stimulation with CRC cell lines resulted in CD107a-expression of 0.6% and 1.2 % in CD4+ T cells from the first expansion phase and in a range of 0.8% and 2.0% in CD8+ T cells (w/o stimulation: 1.0%–1.1% and 0.9%–1.1 %). In the second phase, stimulation by CRC cell lines led to 0.7%–2.5% CD107a+ CD4+ T cells and 0.9%–2.3% CD107a + CD8+ T cells (w/o stimulation: 0.9%–1.1% and 1.0%–1.1%). Analysis of first-phase TILs revealed 0.5%–1.3% IFNγ + CD4+ T cells and 0.5%–2.3% IFNγ + CD8+ T cells (without stimulation: 1.0%–1.1% and 0.9%–1.1%). At the end of the second phase, 0.5%–1.6% of IFNγ+ CD4+ and 0.8%– 2.2% of IFNγ + CD8+ T cells (without stimulation: 1.0%–1.3% and 1.0%, respectively) were measured. The third molecule, TNFα, was expressed in 0.8%–1.7% first-phase CD4+ cells and 0.7%–2.6% first-phase CD8+ cells (without stimulation: 0.5%–1.1% and 0.8%–1.0 %). After the second phase, 0.7%–2.6% of CD4+ T cells and 0.8%–2.7% of CD8+ T cells were measured positive for TNFα (without stimulation: 0.2%–1.0% and 0.1%–1.1%, respectively).
In summary, co-culturing of TIL and CRC cell lines showed no degranulation and cytokine expression effects after 6 h of stimulation. Only the co-treatment with CRC-cell line plus PMA caused increase of CD107a, IFNγ and TNFα expression (supplementary Figure 2, supplementary Figure 3). A total of 2.7%–22.3% of CD4+ and 22.8%–50.3% of CD8+ first-phase T cells expressed CD107a following co-stimulation of CRC-cell line plus PMA (without stimulation: 1.0%–1.1% and 0.9%–1.1%, respectively). Second phase T-cell stimulation resulted in 5.8%–34.8% CD107a + CD4+ and 35.0%–69.2% CD107a+ CD8+ T cells (without stimulation: 0.9%–1.1% and 1.0%–1.1%). Measurement of first-phase TILs revealed 0.4%–12.1% IFNγ + CD4+ T cells and 2.8%–51.0% IFNγ + CD8+ T cells (without stimulation: 1.0%–1.1% and 0.9%–1.1%, respectively). At the end of the second phase, 0.8%–22.0% of IFNγ + CD4+ and 5.1%–56.3% of IFNγ + CD8+ T cells (without stimulation: 1.0%–1.3% and 1.0%) were found. TNFα was expressed in 8.0%–39.6% first-phase CD4+T cells and 11.7%–57.4% first-phase CD8+T cells (without stimulation: 0.5%–1.1% and 0.8%–1.0%, respectively). In the second phase, TNFα+ CD4+ T cells were measured in a range of 11.1%–55.3% and TNFα+ CD8+ T cells in a range of 11.1%–70.2% after co-stimulation of the CRC cell line plus PMA (without stimulation: 0.2%–1.0% and 0.1%–1.1%, respectively) (supplementary Figure 2, supplementary Figure 3).
Quantitative cytokine assay
The analyses of intracellular cytokine expression were extended by T-cell culture supernatant quantification (both IFNγ and TNFα after CRC-unspecific stimulation by anti-CD3 antibody and PMA (Figure 4, supplementary Table 2). The cytokine levels found in cell culture supernatants after T-cell stimulation mirror the findings of the intracellular cytokine staining protocol. Stimulation by anti-CD3 antibody caused an IFNγ secretion in a range of 120 pg/mL to 2980 pg/mL (control without anti-CD3-stimulation: 54 pg/mL to 265 pg/mL) and a TNFα secretion up to 737 pg/mL (up to 1 pg/mL without stimulation) in cell suspensions, cultivated in the first expansion phase. IFNγ secretion of cells, harvested from second expansion phase, were in a range of 800 pg/mL to 2140 pg/mL (unstimulated: 25 pg/mL to 94 pg/mL) and for TNFα secretion in a range of 183 pg/mL to 578 pg/mL (unstimulated: 0.5 pg/mL to 1.5 pg/mL) in cell suspensions (Figure 4, supplementary Table 2).
Fig. 4Cytokine concentrations (IFNγ, TNFα): TILs were analyzed after stimulation with anti-CD3 Ab. First (A) and second (B) expansion phases were examined. Each bar represents mean value of double measurements (mean ± standard error of the man). The individual values of each tumor can be found in supplementary Table 2.
In the development of new therapeutic strategies for colorectal cancer, it was found that only the less-frequent tumors with dMMR-MSI-H respond to immunotherapy with checkpoint inhibitors [
], ACT using TILs is a reasonable approach that takes advantage of the patient's specific T-cell immune response towards the tumor.
The potential success of ACT depends on both the quantity and quality of expanded TILs from tumor tissue. In terms of quantity, a number of 1 × 1010 expanded TILs is considered an appropriate dose for a therapeutic infusion [
Pörtner, R., Parida, S., Schaffer, C., and Hoffmeister, H. (2018). "Landscape of manufacturing process of ATMP cell therapy products for unmet clinical needs." 2018 May 2; Available from: http://dx.doi.org/10.5772/intechopen.69335.
]. However, in a pilot study using expanded T cells from CRC sentinel lymph nodes for ACT, complete tumor responses were achieved with only 8 × 107 cells in metastatic CRC [
Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy.
In our study, the obtained tumor tissue samples were subjected to pathological screening, in which TILs were examined in H&E and by immunostaining. Pathological screening revealed TIL infiltration rates of 5%–60% and a varying CD4+/CD8+ ratio in favor of CD4+ cells (mean 62/30%). Regardless of these initial infiltration rates, our data show that expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor from surgically resected primary CRC resulted in a relevant number of approximately 2 × 109 cells. There was no correlation between TIL infiltration rates on pathological screening and the number of expanded cells. The second phase can be scaled up to manufacture more than 1010 TILs, using a bioreactor with 5 times more expansion area. TIL expansion was performed in a two-phase process to separate cells from medium and residual tumor tissue at the end of phase 1 to avoid tumor-related inhibitory effects.
We observed a strong dependency of TIL outgrowth on tumor mass during starting period. Starting expansion with a relatively large tumor volume (400 mm3; 50 pieces), we noticed limited cell yield after phase 1. Reducing tumor volume (to 240 mm3 and later to 80 mm3) significantly increased cell yield after phase 1 and resulted in an ACT-relevant number of TILs after phase 2. Therefore, the quantity of 80 mm3 was set as an optimized standard. The reason for the phenomenon described is not clear, but is probably related to tumor-induced immune-inhibitory effects with higher volumes of tumor tissue present.
TILs were manufactured on a platform on which perfusion type bioreactors were operated in an automatically controlled culturing process. The outgrowth of TILs from tumor pieces, activation and long-term expansion was performed in a closed perfusion bioreactor system developed at Zellwerk GmbH and established for many years [
Pörtner, R., Parida, S., Schaffer, C., and Hoffmeister, H. (2018). "Landscape of manufacturing process of ATMP cell therapy products for unmet clinical needs." 2018 May 2; Available from: http://dx.doi.org/10.5772/intechopen.69335.
]. This meander bioreactor is characterized by a directional laminar flow of the medium, which allows homogeneous distribution of nutrients and gases over the cultivation period and minimizes disruption of cell–cell and cell–surface contact.
Regarding the subtypes of expanded TILs, CD8+ T cells are the most important cell population for the anti-tumor response [
Adoptive transfer of tumor-primed, in vitro-activated, CD4+ T effector cells (TEs) combined with CD8+ TEs provides intratumoral TE proliferation and synergistic antitumor response.
In our study, the CD4+/CD8+ ratio constantly shifted towards CD8+ cells during TIL expansion, resulting in about 65% after phase 1 and about 86% CD8+ cells after phase 2. This is in contrast to other studies, which reported that a lower proportion of CD8+ cells were expanded from GI tumors compared to melanoma, in which >70% CD8+ cells are routinely found [
Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy.
]. However, in these studies, TILs were mainly expanded from metastases of GI tumors. It remains unclear whether the different tumor material or the expansion process account for the different results.
In this study, double-positive (CD4+ CD8+) cells were found in a small proportion of approximately 2.4% of TILs after expansion phase 1 and 0.8% after phase 2, respectively. Tumor reactivity was noted for these double-positive TILs in CRC. However, because the frequency of double-positive TILs is increased in metastatic CRC, it has been postulated that this subset of TILs may play a role in the metastatic process by downregulating the immune response to the tumor [
]. The significance of the double-positive cells among expanded TILs is not yet determined.
Further characterization of ACT-relevant TIL-subtypes includes markers for antigen experience and differentiation of effector (Teff) and regulatory T cells (Treg). Previous preclinical and clinical studies of ACT have mostly used unselected T cells, including cytotoxic, helper, and even immunosuppressive Treg cells [
]. More precise characterization and selection of the T cells used could be a promising approach to improve efficacy and safety of ACT.
It has been shown that a high Teff/Treg ratio is essential for effective immunosurveillance of GI tumors and is therefore target for an ACT-relevant TIL population [
]. The T cells found in the inhibitory tumor microenvironment are predominantly Treg cells, but appear to have a plastic phenotype that can be polarized toward effector function upon expansion in IL-2 [
Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy.
CD45RO has been shown to be a suitable marker for (tumor antigen)-activated CD8+ T effector cells, which have the potential to elicit an anti-tumor immune response [
]. Therefore, aiming for a high proportion of CD45RO+ T cells could increase effectiveness of TILs in the context of ACT-approaches.
CCR7 expression divides memory T cells into two functional subsets. CCR7− memory cells have direct effector function and are therefore considered effector memory T cells (TEM). CCR7+ cells are central memory T cells (TCM) that express lymph node homing receptors but have no immediate effector function [
In the current study, the phenotype of CD8+ effector memory cells (CD45RO+/CCR7−) accounted for approximately 60% of TILs after expansion phase 1 and 73% of TILs after phase 2, respectively. This suggests that this cell population could have a long-term cytotoxic effect against tumor cells.
To investigate the functional potential of the obtained TILs upon nonspecific stimulation, we chose a time frame of 6 h of stimulation suitable for testing also in the context of further clinical use, both with regard to quantification of intracellular cytokine expression as well as for quantification of degranulation in the supernatant.
Anti-CD3 stimulation and intracellular cytokine staining revealed CD8+ IFNγ+ cells in 3% of TILs in expansion phase 1 and 4.2% of TILs in phase 2, respectively. These data appear to be small compared with reports in the literature. It should be noted, however, that the duration of stimulation in these studies usually ranged from 12 hours up to several days [
Adoptive transfer of tumor-primed, in vitro-activated, CD4+ T effector cells (TEs) combined with CD8+ TEs provides intratumoral TE proliferation and synergistic antitumor response.
]. PMA stimulation lasting 6 hours resulted in CD8+ IFNγ+ cells in 50% of TILs harvested in phase 1 and in 55% of TILs in expansion phase 2.
In addition, quantification of degranulation showed relevant cytokine secretion in the supernatant upon both anti-CD3 and PMA stimulation for 6 hours. Overall, the functional potential of TILs expanded in our study was confirmed upon nonspecific stimulation.
In conclusion, a reliable, standardized bioreactor process for TIL expansion from primary CRC tissue samples was established, and a relevant number of TILs of an ACT-relevant subtype with functional potential could be obtained (the entire process, exemplified in supplementary Figure 4 and supplementary Figure 5). In this process, the Zellwerk ZRP bioreactor enabled a high degree of standardization of immune cell mass production in a closed environment under constant conditions.
The present study investigated the feasibility of expanding TIL from resected primary CRC tumors, which were predominantly mismatch repair-proficient-microsatellite instability-low (pMMR-MSI-L), and established high-volume immune cell generation also from these tumors, which are not sensitive to checkpoint inhibitor immunotherapy approaches. Therefore, ACT options using TILs could be an approach to harness an anti-tumor immune response for this subset of CRC as well.
However, this study did not investigate the cytotoxic functional potential of expanded TILs when stimulated with autologous tumors cells. At the time of the investigation, autologous tumor material was no longer available. As an approximation, we examined the functional potential of TILs when challenged with a series of commercially available CRC tumor cell lines. However, degranulation and intracellular cytokine expression (IFNγ, TNFα) after short-time challenge with CRC cell lines did not demonstrate the cytotoxic potential of the expanded TILs. When co-cultured with PMA as a stimulation control, TILs were shown to have functional potential (compared to observations in assays without tumor cell lines). However, the CRC cell lines used in this study did not achieve specific stimulation of expanded TILs. It has been previously reported that GI tumors have unique mutations and antitumor T-cell responses are directed to neoantigens and cryptic peptides specific to each individual patient [
Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy.
]. Therefore, commercially available colon cancer cell lines are probably not a good model for demonstrating specific cytotoxicity of expanded TILs. In further studies, we will evaluate the cytotoxic function of expanded TILs when stimulated with either cryopreserved autologous tumor cell suspensions, tumor cell-derived material or autologous cell lines.
Furthermore, it will be the next step to test TIL populations expanded from patients using the method described here in a broader range of in vitro experiments to study a wide range of TIL function modifiers, e.g., polyunsaturated fatty acids and lipid mediators that have a role in colon tumorigenesis [
]. In addition, factors such as preoperative immune-nutrition and gut microbiota influence the composition and function of TILs in colorectal cancer and are therefore a promising subject for further studies [
Based on these planned in vitro cytotoxic function experiments of the expanded TILs, future in vivo studies could be envisaged. Notably, because the complex tumor microenvironment may affect TIL function in vivo, in vitro data can only serve as an approximation. The main obstacles to ACT in vivo appear to be short-term persistence and poor trafficking of immune cells within the immunosuppressive tumor microenvironment, anergy and poor and insufficient proliferation of immune cells [
], effects that can only be evaluated and understood in vivo.
We focused on CRC primary tumors when establishing the TIL expansion process described here in order to exclude any influences of previous therapies (radiation, chemotherapy, immunotherapy). However, given that ACT approaches might primarily be ultima ratio treatment options in heavily pretreated patients, primary tumor tissue is unlikely to be the material of choice for TIL expansion in these cases. Therefore, in a next step we will investigate whether the promising results of TIL expansion from CRC primary tumors presented here also apply to TIL expansion from CRC metastases.
Declaration of competing interests
The authors have no commercial, proprietary or financial interest in the products or companies described in this article.
Authors contributions
HCA, DG, and JS collected and analyzed the data and wrote main parts of the manuscript. HCA, SG, JS, and FL collected and analyzed the tumor samples. HCA, SG, HH, WD, K-HW designed the study and completed the manuscript. DG, HH, and JS expanded and provided the TILs for the experiments. All authors meet the criteria of the International Committee of Medical Journal Editors (ICMJE) regarding the definition of authorship and have approved the final article.
Funding
HH is the CEO of Zellwerk GmbH and has patents (DE102018000561, EP3517600, WO2020078498) for a bioreactor-based expansion process for cells from tumors. DG works for Zellwerk GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Assessing tumor-infiltrating lymphocytes in solid tumors: a practical review for pathologists and proposal for a standardized method from the international immunooncology biomarkers working group: part 1: assessing the host immune response, TILs in invasive breast carcinoma and ductal carcinoma in situ, metastatic tumor deposits and areas for further research.
Hoffmeister, H. (2019). Meander bioreactor and method for the isolation and proliferation of cells from parts from tumors, metastases and other tissues. Germany patent application.
Hoffmeister, H. (2020). Meandering perfusion bioreactor for the differentiation, activation, stimulation and separation of cells. Germany patent application.
Pörtner, R., Parida, S., Schaffer, C., and Hoffmeister, H. (2018). "Landscape of manufacturing process of ATMP cell therapy products for unmet clinical needs." 2018 May 2; Available from: http://dx.doi.org/10.5772/intechopen.69335.
Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy.
Adoptive transfer of tumor-primed, in vitro-activated, CD4+ T effector cells (TEs) combined with CD8+ TEs provides intratumoral TE proliferation and synergistic antitumor response.
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