Good Manufacturing Practice Conditions Research Articles

Clinical-grade human embryonic stem cell–derived mesenchymal stromal cells ameliorate diabetic retinopathy in db/db mice

Background aimsMesenchymal stromal cells (MSCs) hold great promise in the treatment of diabetic retinopathy (DR), as evidenced by increasing preclinical and clinical studies. However, the absence of standardized and industrialized clinical-grade donor cells hampers the continued development and large-scale clinical application of MSCs-based therapies for DR. Previously, we have identified a unique population of MSCs generated from a clinical-grade human embryonic stem cell (hESC) line under Good Manufacturing Practice conditions that could be a potential source to address the issues. Here, we investigated the therapeutic potential of the clinical-grade hESC line–derived MSCs (hESC-MSCs) on db/db mice with DR. MethodshESC-MSCs were initially characterized by morphological assessment, flow cytometry analysis and trilineage differentiation assays. These cells (5 × 106 cells) were then transplanted intravenously into 12-week-old db/db mice via tail vein, with phosphate-buffered saline transplantation and untreated groups used as controls. The retinal alterations in neural functions and microvascular perfusions, and inflammatory responses in peripheral blood and retina were evaluated at 4 and 6 weeks after transplantation using electroretinography, optical coherence tomography angiography and flow cytometry, respectively. Body weight and fasting blood glucose (FBG) levels were also measured to investigate their systemic implications. ResultsCompared with controls, intravenous transplantation of hESC-MSCs could significantly: (i) enhance impaired retinal electroretinography functions (including amplitudes of a-, b-wave and oscillatory potentials) at 4 weeks after transplantation; (ii) alleviate microvascular dysfunctions, especially in the inner retina with significance (including reducing non-perfusion area and increasing vascular area density) at 4 weeks after transplantation; (iii) decrease FBG levels at 4 weeks after transplantation and induce weight loss up to 6 weeks after transplantation and (iv) increase both peripheral blood and retinal interleukin-10 levels at 4 weeks after transplantation and modulate peripheral blood inflammatory cytokines and chemokines levels, such as monocyte chemotactic protein-1, up to 6 weeks after transplantation. ConclusionsThe findings of our study indicated that intravenous transplantation of hESC-MSCs ameliorated retinal neural and microvascular dysfunctions, regulated body weight and FBG and modulated peripheral blood and retinal inflammation responses in a mouse model of DR. These results suggest that hESC-MSCs could be a potentially effective clinical-grade cell source for the treatment of DR.

Good Manufacturing Practice–compliant human induced pluripotent stem cells: from bench to putative clinical products

Background aimsFew human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. MethodsOur standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). ResultsTwo MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). ConclusionsWe successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation.

The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease.

PD-L1 expression levels in mesenchymal stromal cells predict their therapeutic values for autoimmune hepatitis

BackgroundAutoimmune hepatitis is a chronic inflammatory hepatic disorder with no effective treatment. Mesenchymal stromal cells (MSCs) have emerged as a promising treatment owing to their unique advantages. However, their heterogeneity is hampering use in clinical applications.MethodsWharton’s jelly derived MSCs (WJ-MSCs) were isolated from 58 human donors using current good manufacturing practice conditions. Gene expression profiles of the WJ-MSCs were analyzed by transcriptome and single-cell RNA-sequencing (scRNA-seq), and subsequent functional differences were assessed. Expression levels of programmed death-ligand 1 (PD-L1) were used as an indicator to screen WJ-MSCs with varied immunomodulation activities and assessed their corresponding therapeutic effects in a mouse model of concanavalin A-induced autoimmune hepatitis.ResultsThe 58 different donor-derived WJ-MSCs were grouped into six gene expression profile clusters. The gene in different clusters displayed obvious variations in cell proliferation, differentiation bias, trophic factor secretion, and immunoregulation. Data of scRNA-seq revealed four distinct WJ-MSCs subpopulations. Notably, the different immunosuppression capacities of WJ-MSCs were positively correlated with PD-L1 expression. WJ-MSCs with high expression of PD-L1 were therapeutically superior to WJ-MSCs with low PD-L1 expression in treating autoimmune hepatitis.ConclusionPD-L1 expression levels of WJ-MSCs could be regarded as an indicator to choose optimal MSCs for treating autoimmune disease. These findings provided novel insights into the quality control of MSCs and will inform improvements in the therapeutic benefits of MSCs.

Preclinical and dose-ranging assessment of hESC-derived dopaminergic progenitors for a clinical trial on Parkinson's disease

Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.

Breaking Barriers in B-Acute Lymphoblastic Leukemia Treatment: Experience of a Tertiary Hospital with the Use of a Tandem Dual CAR-T 19/22 in Pediatric & AYA Patients

INTRODUCTION Anti-CD19 chimeric antigen receptor (CAR) autologous T-cell therapy tisagenlecleucel (CTL019) has shown efficacy in pivotal trials and real-world data in relapsed/refractory pediatric B-cell precursor acute lymphoblastic leukemia (r/r B-ALL). However, 30-50% of patients relapse, because of CD19 loss in leukemic cells, or persistence/function reduction of CTL019. To improve relapse free survival, multitargeting CAR-T cells, such as bicistronic anti-CD22 and anti-CD19 or single sequential infusion are being developed. We report the safety and efficacy of an in-house manufactured tandem anti-CD19/22 dual CAR-T cells in pediatric, adolescents and young adults (AYA) diagnosed with r/r B-ALL. MATERIAL AND METHODS Eight batches of the tandem anti-CD19/22 dual CAR-T cells were manufactured under Good Manufacturing Practices (GMP) and authorized under a compassionate use program by the Spanish Agency for Medicines and Health (AEMPS). The starting material in all cases was patient´s non mobilized apheresis. After, CD4 + and CD8 + T cells were immune selected, activated with CD3/CD28 beads and transduced with a CD19/CD22 lentiviral vector (from Miltenyi Biotec®). Cell expansion was performed in a CliniMACS Prodigy® device with TexMACs® culture medium supplemented with IL-7 and IL-15. During manufacturing, process and quality controls were carried out and the finished product was infused fresh or cryopreserved. RESULTS A total of 8 patients with a median age of 10.5 (range 6-25) years old received a median of 1 (range 1-3) doses of tandem anti-CD19/22 dual CAR-T cells containing a median of 1.13x106 (range 7.5x105-3x106) cells/kg infused. First 3 patients (37.5%) received cryopreserved product. Two patients (25%) were also diagnosed with Down syndrome (DS). The median time between apheresis and infusion was 10.5 days (range 9-14 ). Five patients (62.5%) received previously tisagenlecleucel and four of them (50%) had also received a hematopoietic stem cell transplantation (HSCT). Leukemia blasts did not express CD19 in 2 patients (25%). Both received tisagenlecleucel previously, and one of them also expressed low levels of CD22. Median pre-infusion minimal residual disease (MRD) was 4.91% (range 0.01-87). Bridging therapy was low dose chemotherapy in 7 patients (87.5%), using inotuzumab in 1 patient. Lymphodepletion regimen was individualized, in 5 patients (62.5%) consisted on fludarabine 30 mg/m2/day for 4 days and cyclophosphamide 600 mg/m2/day for 2 days. Regarding CAR cell product, mean transduction was 41.4 %. Similar CD4 +/CD8 + distribution was seen in most products generated (7/8, 88%), and central memory cells were the main subpopulation found, followed by naïve cells. Most CAR-T cell expansions (88%, median 7, range 7-28) were seen between day +7 and day +21 post-infusion. Regarding toxicity, 7 patients (87.5%) presented cytokine release syndrome (CRS). Three patients (37.5 %) with high pre CAR-T tumor burden (TB) (range 75-87 %) developed CRS grade 3-4 (ASBMT guidelines) followed by ICANS grade 3. On day +28 after infusion, 6 patients (75%) were in complete remission (CR), one of them with positive minimal residual disease (MRD). Two patients died because of disease progression (range 2-4.5 months) and a third one (with DS) because of toxicity before day +28 after CAR-T infusion. Consolidative HSCT was performed in 3 of 6 patients that achieved CR (50%) during the first 90 days after CAR-T cell infusion. During follow up of these transplanted patients (median 8.4 months, range 2.3-12.3), 1 patient (33.3%) relapsed after 11 months. One patient presented central nervous system relapse 1 month after CAR-T infusion and HSCT was performed and with 35 months of follow up, continues alive in CR. The last patient (with DS) relapsed 3 months after infusion and immunotherapy (inotuzumab) has been given in order to perform a second HSCT if CR is achieved. CONCLUSIONS In-house manufacturing of tandem anti-CD19/22 dual CAR-T cells is feasible and reproducible under GMP conditions in an academic institution. Product infusion seems to be well tolerated in most patients, although patients with high TB developed high grade toxicities. Setting bridging therapy, reducing TB and manufacturing time remains a challenge. Our experience suggests the efficacy of its use as a bridge to consolidative HSCT. Nevertheless, a larger number of patients in the context of a clinical trial would be necessary.

Good practices for the automated production of 18F-SiFA radiopharmaceuticals

BackgroundThe positron emitting isotope fluorine-18 (18F) possesses almost ideal physicochemical properties for the development of radiotracers for diagnostic molecular imaging employing positron emission tomography (PET). 18F in its nucleophilic anionic 18F− form is usually prepared by bombarding an enriched 18O water target with protons of various energies between 5 and 20 MeV depending on the technical specifications of the cyclotron. Large thick-target yields between 5 and 14 GBq/µA can be obtained, enough to prepare large batches of radiotracers capable to serve a considerable contingent of patients (50 + per clinical batch). The overall yield of the radiotracer however depends on the efficiency of the 18F labeling chemistry. The Silicon Fluoride Acceptor chemistry (SiFA) has introduced a convenient and highly efficient way to provide clinical peptide-based 18F-radiotracers in a kit-like procedure matching the convenience of 99mTc radiopharmaceuticals.Main bodyA radiotracer’s clinical success primarily hinges on whether its synthesis can be automated. Due to its simplicity, the SiFA chemistry, which is based on isotopic exchange (18F for 19F), does not only work in a manual setup but has been proven to be automatable, yielding large batches of 18F-radiotracers of high molar activity (Am). The production of SiFA radiotracer can be centralized and the radiopharmaceutical be distributed via the “satellite” principle, where one production facility economically serves multiple clinical application sites. Clinically validated tracers such as [18F]SiTATE and [18F]Ga-rhPSMA-7/-7.3 have been synthesized in an automated synthesis unit under good manufacturing practice conditions and used in large patient cohorts. Communication of common guidelines and practices is warranted to further the dissemination of SiFA radiopharmaceuticals and to give easy access to this technology.ConclusionThis current review highlights the most recent achievements in SiFA radiopharmaceutical automation geared towards large batch production for clinical application. Best practice advice and guidance towards a facilitated implementation of the SiFA technology into new and already operating PET tracer production facilities is provided. A brief outlook spotlights the future potential of SiFA radiochemistry within the landscape of non-canonical labeling chemistries.

High-Titer Recombinant Adenovirus 26 Vector GMP Manufacturing in HEK 293 Cells with a Stirred Single-Use Bioreactor for COVID-19 Vaccination Purposes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 virus) pandemic has shown the importance of pursuing various vaccine manufacturing strategies. In the present study, the HEK 293 cells were infected with recombinant adenovirus serotype 26 (rAd26), and the effects of critical process parameters (CPPs) including viable cell density (VCD) at infection time (0.5 × 106, 0.8 × 106, 1.4 × 106, 1.8 × 106, and 2.5 × 106 cells/mL), the multiplicity of infection (MOI) = 3, 6, 9, 12, and 15, and two aeration strategies (high-speed agitation with a sparging system and low-speed agitation with an overlay system) were investigated experimentally. The results of small-scale experiments in 2 L shake flasks (SF 2L) demonstrated that the initial VCD and MOI could affect the cell proliferation and viability. The results at these experiments showed that VCD = 1.4 × 106 cells/mL and MOI = 9 yielded TCID50 /mL = 108.9, at 72 h post-infection (hpi), while the virus titer at VCD = 0.5 × 106 and 0.8 × 106 cells/mL was lower compared to that of VCD = 1.4 × 106 cells/mL. Moreover, our findings showed that VCDs > 1.8 × 106 cells/m with MOI = 9 did not have a positive effect on TCID50 /mL and MOI = 3 and 6 were less efficient, whereas MOI > 12 decreased the viability drastically. In the next step, the optimized CPPs in a small scale were exploited in a 200 L single-use bioreactor (SUB), with good manufacturing practice (GMP) conditions, at RPM = 25 with an overlay system, yielding high-titer rAd26 manufacturing, i.e., TCID50/mL = 108.9, at 72 hpi.

Validation of the media fill method for Cytokine-Induced killer cells manufacturing process

BackgroundMedia fills are used to demonstrate the adequacy of the aseptic conditions of a pharmaceutical production process, according to good manufacturing practice (GMP), using a growth medium in place of the product solution. For advanced therapy medicinal product (ATMP), where the final product consists of viable cells, the media fill is still a challenge, but represents the starting point for process validation.MethodsThe aim of this paper is to describe the media fill test procedure in the context of ATMP manufacturing, in particular of Cytokine-Induced Killer (CIK) cell expansion process under GMP conditions, including quality control tests and environmental monitoring. The media fill test has been designed to cover all the critical steps of the process, including worst cases and deviations.ResultsFrom July 2019 to August 2022, we performed 16 media fill tests. During these years, the media fill protocol has been gradually improved and the worst cases were designed to be closer to reality and occurring cases. Although some deviations occurred, all the media fills performed were compliant.ConclusionsA good media fill design combined with a robust environmental monitoring program provides a high degree of assurance of the microbial safety of ATMPs.

Therapeutic improvement of Parkinson’s disease transplantation therapy through modulating the immune system.

Abstract We report clinically the implantation of patient-derived midbrain dopaminergic progenitor cells (mDAPs), differentiated in vitrofrom autologous iPSCs, in a patient with idiopathic Parkinson's disease (PD). The patient-specific mDAPs were produced under Good Manufacturing Practice conditions and characterized as having the phenotypic properties of substantia nigra pars compacta neurons; testing in a humanized mouse model indicated an absence of immunogenicity to these cells. The cells were implanted into the putamen (left hemisphere followed by right hemisphere, 6 months apart) of a patient with PD, without the need for immunosuppression. Clinical measures of symptoms of PD after surgery stabilized or improved at 18 to 24 months after implantation. Although our previous data (Schweitzer*, Park* et al., NEJM, 382(20) p1926–1932, 2020) showed that using autologous mDAPs can avoid graft rejection, cell survival and functional recovery in the first patient as assessed by 18F-DOPA PET scan analysis and clinical motor assessments was modest, suggesting that survival of mDA neurons may have been suboptimal even in autologous implantation. We have developed a safer and more effective treatment by not only increasing mDA neuron survival by simultaneously controlling the causative immune response, but also by modulating the problematic proliferative property, which is a characteristic of progenitor cells after implantation. In conclusion, our new study suggests that initial, specific mDA neuron survival in these grafts is strongly affected by the host innate immune response, while long-term graft survival is subject to the adaptive immune response, elucidating a two-phase involvement of the immune system for graft survival. This work was supported by NIH grants (NS070577 and OD024622), the Parkinson’s Cell Therapy Research Fund at McLean Hospital and Massachusetts General Hospital, and the Masson Family Endowed Scholar in Neurosurgery, and the George A. Lopez, MD Endowed Chair in Neurosurgery.

How to Develop a Rare Disease Treatment

How to Develop a Rare Disease Treatment

P770 Autologous regulatory T cell transfer in patients with refractory ulcerative colitis: Interim report of a phase 1, dose-escalation trial

Abstract Background Ulcerative colitis (UC) is marked by impaired mucosal homeostasis with predominance of intestinal effector T cells and insufficient expansion of mucosal regulatory T cells (Tregs), thereby providing a scientific rationale for Treg-based immunotherapy in UC. Methods We developed a protocol to generate large numbers of autologous CD25+ cells under aegis of IL-2, rapamycin and anti-CD3/anti-CD28 expander beads ex vivo from CD25+ precursors derived from peripheral blood cells, intended for clinical use under good manufacturing practice (GMP) conditions. We initiated a single-centre, open-label, fast-track dose-escalation, phase-1 clinical trial with a single adoptive transfer of autologous ex vivo expanded CD4+CD25+CD127−/lo Tregs in patients with refractory UC. The primary objective of the trial was to define safety and the maximum tolerable dose of a single intravenous administration of autologous expanded Tregs according to the fast-track dosing principle. Adoptive transfer was escalated from the starting dose of 0.5×106 Tregs/kg body weight to the next dose level (1×106, 2×106, 5×106 and 10×106 Tregs/kg body weight) in each consecutive patient, respectively, if no dose-limiting toxicity occurred. We here report interim study data of the first 8 patients that received Treg transfer and were followed up over 12 weeks. There were no changes in concomitant medication during the study period. Results The 8 patients received 0.5 x106 (n=1), 1×106 (n=1), 2×106 (n=1), 2.8x106 (n=1), 5×106 (n=1) and 10×106 (n=3) Treg/kg body weight, respectively. The patients did not show any dose-limiting toxicity and there were no adverse events related to Treg transfer. 87.5% (7/8) of patients had previously received treatment with at least 2 different substance classes of advanced therapies, such as biologics and JAK inhibitors and the mean Mayo Score (MS) at baseline was 9.1±1.6. The mean modified MS (mMS) dropped significantly from 6.6±1.2 at baseline to 4.5±2.2 at week 4 (p=0.006) and 4.6±2.0 at week 12 (p=0.01). There was reduction of mean faecal Calprotectin levels from 1708±397 µg/g at baseline, to 1411±471 µg/g at week 4 and 919±332 µg/g at week 12. Clinical response was reached by 62.5% (5/8) of patients at week 4 and also week 12. Clinical response predominantly occurred in patients receiving the highest number of 10×106 Tregs/kg body weight. Clinical remission was achieved by 12.5% (1/8) of patients at week 4 and also week 12. Conclusion Autologous regulatory T cell transfer was well tolerated up to the maximal tested dose of 10×106 Tregs/kg body weight. Study results suggest that adoptively transferred polyclonal Tregs might be effective in refractory ulcerative colitis, warranting further clinical studies.

Manufacturing and validation of Good Manufacturing Practice–compliant regulatory dendritic cells for infusion into organ transplant recipients

Background aimsRegulatory (or “tolerogenic”) dendritic cells (DCregs) are a highly promising, innovative cell therapy for the induction or restoration of antigen-specific tolerance in immune-mediated inflammatory disorders. These conditions include organ allograft rejection, graft-versus-host disease following bone marrow transplantation and various autoimmune disorders. DCregs generated for adoptive transfer have potential to reduce patients’ dependence on non-specific immunosuppressive drugs that can induce serious side effects and enhance the risk of infection and certain types of cancer. Here, our aim was to provide a detailed account of our experience manufacturing and validating comparatively large numbers of Good Manufacturing Practice–grade DCregs for systemic (intravenous) infusion into 28 organ (liver) transplant recipients and to discuss factors that influence the satisfaction of release criteria and attainment of target cell numbers. ResultsDCregs were generated in granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 from elutriated monocyte fractions isolated from non-mobilized leukapheresis products of consenting healthy adult prospective liver transplant donors. Vitamin D3 was added on day 0 and 4 and IL-10 on day 4 during the 7-day culture period. Release and post-release criteria included cell viability, purity, phenotype, sterility and functional assessment. The overall conversion rate of monocytes to DCregs was 28 ± 8.2%, with 94 ± 5.1% product viability. The mean cell surface T-cell co-inhibitory to co-stimulatory molecule (programmed death ligand-1:CD86) mean fluorescence intensity ratio was 3.9 ± 2.2, and the mean ratio of anti-inflammatory:pro-inflammatory cytokine product (IL-10:IL-12p70) secreted upon CD40 ligation was 60 ± 63 (median = 40). The mean total number of DCregs generated from a single leukapheresis product (n = 25 donors) and from two leukapheresis products (n = 3 donors) was 489 ± 223 × 106 (n = 28). The mean total number of DCregs infused was 5.9 ± 2.8 × 106 per kg body weight. DCreg numbers within a target cell range of 2.5–10 × 106/kg were achieved for 25 of 27 (92.6%) of products generated. ConclusionsHigh-purity DCregs meeting a range of quality criteria were readily generated from circulating blood monocytes under Good Manufacturing Practice conditions to meet target cell numbers for infusion into prospective organ transplant recipients.

Formulation of a kit under Good Manufacturing Practices (GMP) for preparing [111In]In-BnDTPA-trastuzumab-NLS injection: a theranostic agent for imaging and Meitner-Auger Electron (MAE) radioimmunotherapy of HER2-positive breast cancer

Background111In[In]-BnDTPA-trastuzumab-NLS is a radiopharmaceutical with theranostic applications for imaging and Meitner-Auger electron (MAE) radioimmunotherapy (RIT) of HER2-positive breast cancer (BC). Nuclear localization sequence (NLS) peptides route the radiopharmaceutical to the nucleus of HER2-positive BC cells following receptor-mediated internalization for RIT with subcellular range MAEs. The γ-photons emitted by 111In permit tumour imaging by SPECT. Our aim was to formulate a kit under Good Manufacturing Practices conditions to prepare 111In[In]-BnDTPA-trastuzumab-NLS injection for a first-in-human clinical trial.ResultsTrastuzumab was derivatized with p-SCN-BnDTPA to introduce Bn-DTPA for complexing 111In, then modified with maleimide groups for conjugation to the thiol on cysteine in NLS peptides [CGYGPKKKRKVGG]. BnDTPA-trastuzumab-NLS (5 mg in 1.0 mL of 0.05 M ammonium acetate buffer, pH 5.5) was dispensed into unit dose sterile glass vials to produce kits for labeling with 100–165 MBq of 111In[In]Cl3. The kits met specifications for protein concentration (4.5–5.5 mg/mL), volume (0.95–1.05 mL), pH (5.5–6.0), appearance (clear, pale-yellow, particulate-free), BnDTPA substitution level (2.0–7.0 BnDTPA/trastuzumab), purity and homogeneity (SDS-PAGE and SE-HPLC), 111In labeling efficiency (> 90%), binding to HER2-positive SK-BR-3 human breast cancer cells (Ka = 1–8 × 108 L/mmol; Bmax = 0.5–2 × 106 sites/cell), NLS peptide conjugation (upward band shift on SDS-PAGE), sterility (USP Sterility Test) and endotoxins (USP Bacterial Endotoxins Test). 111In-BnDTPA-trastuzumab-NLS injection met specifications for pH (5.5–6.5), radiochemical purity (≥ 90%), radionuclide purity (≥ 99%), appearance (clear, colourless, particle-free) and sterility (retrospective USP Sterility Test). Kits were stable stored at 2–8 °C for up to 661 days (d) meeting all key specifications. Protein concentration remained within or just slightly greater than the specification for up to 139 d. 111In[In]-BnDTPA-trastuzumab-NLS injection was stable for up to 24 h. An expiry of 180 d was assigned for the kits and 8 h for the final radiopharmaceutical.ConclusionA kit was formulated under GMP conditions for preparing 111In[In]-BnDTPA-trastuzumab-NLS injection. This radiopharmaceutical was safely administered to 4 patients with HER2-positive BC to trace the uptake of trastuzumab into brain metastases before and after MRI-guided focused ultrasound (MRIg-FUS) by SPECT imaging.

Rapidly accelerated development of neutralizing COVID-19 antibodies by reducing cell line and CMC development timelines.

Since the Coronavirus Disease 2019(COVID-19) outbreak, unconventional cell line development (CLD) strategies have been taken to enable development of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)-neutralizing antibodies at expedited speed. We previously reported a novel chemistry, manufacturing, and control (CMC) workflow and demonstrated a much-shortened timeline of 3-6 months from DNA to investigational new drug (IND) application. Hereafter, we have incorporated this CMC strategy for many SARS-CoV-2-neutralizing antibody programs at WuXi Biologics. In this paper, we summarize the accelerated development of a total of seven antibody programs, some of which have received emergency use authorization approval in less than 2 years. Stable pools generated under good manufacturing practice (GMP) conditions consistently exhibited similar productivity and product quality at different scales and batches, enabling rapid initiation of phase I clinical trials. Clones with comparable product quality as parental pools were subsequently screened and selected for late-stage development and manufacturing. Moreover, a preliminary stability study plan was devised to greatly reduce the time required for final clone determination and next-generation sequencing-based viral testing was implemented to support rapid conditional release of the master cell bankfor GMP production. The successful execution of these COVID-19 programs relies on our robust, fit for purpose, and continuously improving CLD platform. The speed achieved for pandemic-related biologics development may innovate typical biologics development timelines and become a new standard in the industry.

A pragmatic authenticity assessment of lemon (Citrus limon [L.] Burm.f.) juices by its profile of coumarins, psoralens, and polymethoxyflavones

Coumarins, psoralens, and polymethoxyflavones have long been described as authenticity markers in lemon (Citrus limon [L.] Burm.f.) juices. However, the lack of quantitative concentration limits for these markers hampers the differentiation of natural variability and acceptable technical process variations from inacceptable lacks in good manufacturing practice (GMP) or even deliberate food fraud by, e.g., the addition of lime (Citrus × aurantifolia [Christm.] Swingle; Citrus × latifolia [Yu.Tanaka]) juice. In this study, a total of 139 lemon and lime samples, including fruits, juices, juice concentrates, and peel oils from eleven countries and covering all usual processing methods on the market, were analysed using three different liquid chromatographic methods of three different labs. First, we confirmed the considered analytical approaches to yield comparable results and analysed the results in detail per product type and processing technology. After carefully eliminating samples prone to authenticity doubts, we used the remaining dataset (125 samples) for establishing maximum concentration limits for coumarins, psoralens, and polymethoxyflavones for the differentiation of authentic lemon juices produced under GMP conditions, i.e., compliant with the AIJN Code of Practice, from adulterated products made with inacceptable procedures such as excessive co-extraction of flavedo or the admixture of non-lemon Citrus species, particularly lime.

Toxicity Assessment of [177Lu]Lu-iFAP/iPSMA Nanoparticles Prepared under GMP-Compliant Radiopharmaceutical Processes.

The fibroblast activation protein (FAP) is heavily expressed in fibroblasts associated with the tumor microenvironment, while the prostate-specific membrane antigen (PSMA) is expressed in the neovasculature of malignant angiogenic processes. Previously, we reported that [177Lu]lutetium sesquioxide-iFAP/iPSMA nanoparticles ([177Lu]Lu-iFAP/iPSMA) inhibit HCT116 tumor progression in mice. Understanding the toxicity of [177Lu]Lu-iFAP/iPSMA in healthy tissues, as well as at the tissue and cellular level in pathological settings, is essential to demonstrate the nanosystem safety for treating patients. It is equally important to demonstrate that [177Lu]Lu-iFAP/iPSMA can be prepared under good manufacturing practices (GMP) with reproducible pharmaceutical-grade quality characteristics. This research aimed to prepare [177Lu]Lu-iFAP/iPSMA under GMP-compliant radiopharmaceutical processes and evaluate its toxicity in cell cultures and murine biological systems under pathological environments. [177Lu]Lu2O3 nanoparticles were formulated as radiocolloidal solutions with FAP and PSMA inhibitor ligands (iFAP and iPSMA), sodium citrate, and gelatin, followed by heating at 121 °C (103-kPa pressure) for 15 min. Three consecutive batches were manufactured. The final product was analyzed according to conventional pharmacopeial methods. The Lu content in the formulations was determined by X-ray fluorescence. [177Lu]Lu-iFAP/iPSMA performance in cancer cells was evaluated in vitro by immunofluorescence. Histopathological toxicity in healthy and tumor tissues was assessed in HCT116 tumor-bearing mice. Immunohistochemical assays were performed to corroborate FAP and PSMA tumor expression. Acute genotoxicity was evaluated using the micronuclei assay. The results showed that the batches manufactured under GMP conditions were reproducible. Radiocolloidal solutions were sterile and free of bacterial endotoxins, with radionuclidic and radiochemical purity greater than 99%. The lutetium content was 0.10 ± 0.02 mg/mL (0.9 GBq/mg). Significant inhibition of cell proliferation in vitro and in tumors was observed due to the accumulation of nanoparticles in the fibroblasts (FAP+) and neovasculature (PSMA+) of the tumor microenvironment. No histopathological damage was detected in healthy tissues. The data obtained in this research provide new evidence on the selective toxicity to malignant tumors and the absence of histological changes in healthy tissues after intravenous injection of [177Lu]Lu-iFAP/iPSMA in mammalian hosts. The easy preparation under GMP conditions and the toxicity features provide the added value needed for [177Lu]Lu-iFAP/iPSMA clinical translation.

Individualised immunosuppression with intravenously administered donor-derived modified immune cells compared with standard of care in living donor kidney transplantation (TOL-2 Study): protocol for a multicentre, open-label, phase II, randomised controlled trial

IntroductionDonor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in...

Successful Generation of CD19 Chimeric Antigen Receptor T Cells from Patients with Advanced Systemic Lupus Erythematosus

Although it has been shown that the production of functional chimeric antigen receptor T cells is feasible in patients with B-cell malignancies, it is currently unclear whether sufficient amounts of functional autologous CAR T cells can be generated from patients with autoimmune diseases. Intrinsic T-cell abnormalities and T-cell-targeted immune suppression in patients with autoimmunity may hamper the retrieval of sufficient T cells and their transduction and expansion into CAR T cells. Patients with active systemic lupus erythematosus (SLE) underwent leukapheresis after tapering glucocorticoids and stopping T-cell-suppressive drugs. This material was used as source for manufacturing anti-CD19 CAR T-cell products (CAR) in clinical scale. Cells were transduced with a lentiviral anti-CD19 CAR vector and expanded under good manufacturing practice (GMP) conditions using a closed, semi-automatic system. Functionality of these CAR T cells derived from autoimmune patient cells was tested in vitro. Six SLE patients were analyzed. Leukapheresis could be successfully performed in all patients yielding sufficient T-cell numbers for clinical scale CAR T-cell production. In addition, CAR T cells showed high expansion rates and viability, leading to CAR T cells in sufficient doses and quality for clinical use. CAR T cells from all patients showed specific cytotoxicity against CD19+ cell lines in vitro. GMP grade generation of CD19 CAR T-cell products suitable for clinical use is feasible in patients with autoimmune disease.

Marrow changes and reduced proliferative capacity of mesenchymal stromal cells from patients with “no-option” critical limb ischemia; observations on feasibility of the autologous approach from a clinical trial

Background aimsApproximately 1 in 3 patients with critical limb ischemia (CLI) are not suitable for surgical or endovascular revascularization. Those “no-option” patients are at high risk of amputation and death. Autologous bone marrow mesenchymal stromal cells (MSCs) may provide a limb salvage option. In this study, bone marrow characteristics and expansion potentials of CLI-derived MSCs produced during a phase 1b clinical trial were compared with young healthy donor MSCs to determine the feasibility of an autologous approach. Cells were produced under Good Manufacturing Practice conditions and underwent appropriate release testing. MethodsFive bone marrow aspirates derived from patients with CLI were compared with six young healthy donor marrows in terms of number of colony-forming units–fibroblast (CFUF) and mononuclear cells. The mean population doubling times and final cell yields were used to evaluate expansion potential. The effect of increasing the volume of marrow on the CFUF count and final cell yield was evaluated by comparing 5 CLI-derived MSCs batches produced from a targeted 30 mL of marrow aspirate to five batches produced from a targeted 100 mL of marrow. ResultsCLI-derived marrow aspirate showed significantly lower numbers of mononuclear cells with no difference in the number of CFUFs when compared with healthy donors’ marrow aspirate. CLI-derived MSCs showed a significantly longer population doubling time and reduced final cell yield compared with young healthy donors' MSCs. The poor growth kinetics of CLI MSCs were not mitigated by increasing the bone marrow aspirate from 30 to 100 mL. ConclusionsIn addition to the previously reported karyotype abnormalities in MSCs isolated from patients with CLI, but not in cells from healthy donors, the feasibility of autologous transplantation of bone marrow MSCs for patients with no-option CLI is further limited by the increased expansion time and the reduced cell yield.