Chimeric antigen receptor (CAR) T cell therapy has effectively complemented the treatment of advanced relapsed and refractory hematological cancers. The remarkable achievements of CD19- and BCMA-CAR ...T therapies have raised high expectations within the fields of hematology and oncology. These groundbreaking successes are propelling a collective aspiration to extend the reach of CAR therapies beyond B-lineage malignancies. Advanced CAR technologies have created a momentum to surmount the limitations of conventional CAR concepts. Most importantly, innovations that enable combinatorial targeting to address target antigen heterogeneity, using versatile adapter CAR concepts in conjunction with recent transformative next-generation CAR design, offer the promise to overcome both the bottleneck associated with CAR manufacturing and patient-individualized treatment regimens. In this comprehensive review, we delineate the fundamental prerequisites, navigate through pivotal challenges, and elucidate strategic approaches, all aimed at paving the way for the future establishment of multitargeted immunotherapies using universal CAR technologies.
Recently, a static in vitro digestion (IVD) protocol was published by Minekus and coworkers (Minekus et al., 2014) within the COST INFOGEST network. The protocol, concentrating on physiological ...enzyme activities had the main goal to improve the comparability of experimental data between labs. The protocol was validated in several inter-laboratory studies using skim milk powder (SMP) and indeed demonstrated improved harmonization compared with previous experiments with individual IVD protocols (Egger et al., 2016). Although the enzyme activities and salt concentrations of the harmonized protocol are based on available human in vivo data, confirmation of the protocol's physiological relevance has been lacking until now. The main goal of the study was therefore to compare the harmonized IVD protocol with data from in vivo digestion. Towards this aim, an in vivo pig experiment with the same SMP as used for the validation of the IVD protocol was performed followed by a comparison of protein hydrolysis between in vivo and in vitro results. Protein hydrolysis at different levels was analyzed with gel electrophoresis, mass spectrometry, high performance liquid chromatography, and spectrophotometric o-phthaldialdehyde determination of free amino acids. Principle component analysis was used for graphical data comparison.
Milk proteins detected after gastric IVD corresponded to gastric and duodenal in vivo samples and intestinal IVD samples corresponded to distal jejunal in vivo samples. Peptides identified after the gastric phase of IVD, correlated with in vivo gastric samples (r=0.8) and intestinal IVD peptides correlated best with in vivo samples collected from the median jejunum (r=0.57). Free amino acids were in both systems mainly released during the intestinal phase of digestion. Protein hydrolysis in the harmonized IVD was similar to in vivo protein hydrolysis in pigs at the gastric and intestinal endpoints. Therefore, the harmonized static in vitro protocol is suited to study protein hydrolysis at these endpoints.
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•Resistance of lactoglobulin in the gastric step and no intact caseins visible in the intestinal phases in both systems.•Peptide patterns of digested SMP were similar between in vitro and in vivo digestion at the gastric and intestinal endpoints.•In vivo as well as in vitro free amino acids are mainly released during the intestinal phase of digestion.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Notably absent in healthy tissues, or present at inconsequential levels, CTAs are a panel of unique, non-mutated tumour-associated targets for transgenic T-cell receptor-based immunotherapies.2 The ...targeting of CTAs with transgenic T-cell receptors relies exclusively on the intricate interplay of the complex intracellular protein production, the degradation machinery, and the subsequent presentation of its fragments as peptides by the corresponding HLA type. ...because transgenic T-cell receptor-based immunotherapies targeting CTAs are not contingent on missense mutations, it is evident that HLA restriction remains the primary constraint in the broad implementation of transgenic T-cell-based therapies.3 Consequently, one clinically effective transgenic T-cell receptor therapy product can be used universally in patients with different tumour entities that express the respective CTA and share the matching HLA type.2,4 In The Lancet, Sandra P D'Angelo and colleagues report on the interim outcome of the internationally recruiting single-arm phase 2 trial SPEARHEAD-1, using the affinity-optimised transgenic MAGE-A4 T-cell receptor-based autologous T-cell therapeutic afamitresgene autoleucel (afami-cel) for the treatment of patients with advanced or metastatic or unresectable synovial sarcoma or myxoid round cell liposarcoma.4 The trial reports on the clinical findings of selected HLA-A*02-positive patients (44 patients with cytogenetically confirmed synovial sarcoma and eight with myxoid round cell liposarcoma) with confirmed MAGE-A4 expressing advanced or metastatic synovial sarcoma and myxoid round cell liposarcoma with dismal prognosis. The side-effect profile was acceptable with manageable grade 1–2 cytokine release syndrome in 37 (71%) of 52 patients (one grade 3 event) and severe haematotoxicity attributed to the fludarabine and cyclophosphamide conditioning with the upside of improved transgenic T-cell receptor-positive T-cell persistence.5 The primary endpoint of overall response rate was 37% (19 of 52; 95% CI 24–51) of heavily pre-treated patients with synovial sarcoma, including durable responses per Response Evaluation Criteria in Solid Tumours (version 1.1). Despite the relatively prevalent allele frequency of HLA-A*02 across global populations (ranging from 17% to 37% in the tested ethnic groups),7 a substantial number of critically ill patients with cancer unfortunately faced exclusion during the patient selection process due to mismatched HLA types. ...it is noteworthy that approximately only 60% of HLA-A*02-positive patients exhibited adequate MAGE-A4 expression surpassing the predefined threshold, thereby qualifying for inclusion in the trial.4 Nevertheless, individuals meeting the eligibility criteria had considerable treatment benefits from the remarkably effective transgenic T-cell receptor therapy, with a commendable benefit–cost ratio amid the challenging treatment situation faced by patients with advanced refractory cancer with synovial sarcoma and myxoid round cell liposarcoma. Enhancing our understanding of the manufacturing protocol, product composition, and functional data pertaining to transgenic T-cell receptor expression levels, T-cell immunophenotyping, and T-cell exhaustion, along with a focused exploration of the phenotypic and functional characteristics of CD4+ transgenic T-cell receptor-positive T cells, stands as pivotal in optimising the potential of HLA class I targeted transgenic T-cell receptor-based therapies. ...physicians urgently need to further develop novel and complementary therapeutic modalities for patients with advanced synovial sarcoma and myxoid round cell liposarcoma, addressing the current scarcity of treatment options.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Within the context of maximizing the use of dietary phosphorus, a growing-finishing pig study was conducted to determine the optimal total dietary calcium (Ca) to digestible phosphorus (dP) ratio and ...to verify the possibility of mineral phosphate removal during the finishing period on growth performance and mineral status. The potential for replacing chemical and mechanical bone properties by dual energy X-ray absorptiometry (DXA) measures on non-dissected feet was also verified. Three Ca to dP ratios (2.2:1, 2.5:1 and 2.8:1) within two dP levels (P+, P-) were fed during 91 days to 84 pigs. The grower and finisher P+ diets contained 3.0 and 2.4 and P- diets contained 2.5 and 1.7 g dP/kg, respectively. Growth performance and blood serum mineral content were independent of treatments, except that 2.2:1 impaired finisher feed conversion ratio compared to 2.5:1 and 2.8:1. Urinary P concentration increased by 37% in 2.2:1 compared to 2.5:1 and 2.8:1. Maximal load on bone and DXA mineral density were reduced in 2.2:1 compared to 2.8:1. Bone ash and volumetric density were reduced in 2.2:1 and 2.5:1 compared to 2.8:1. Diet P- reduced bone ash, maximal load, volumetric density and DXA bone mineral content and density. No interaction was observed between Ca and dP level. Therefore, 2.2:1 was insufficient for an efficient metabolic use of P, 2.5:1 was sufficient to maximize growth performance and 2.8:1 further improved bone mineralization. Increasing dietary Ca did not impair bone zinc content. Diets P- without supplemented mineral phosphates during the finisher period resulted, per pig, in a decrease of its use by 65% and of the calculated P excretion by 41%, without impaired growth performance. Finally, DXA data responded to dietary treatments as did labor intensive chemical and mechanical bone properties.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Investigations of neuromuscular control of voice production have primarily focused on the roles of muscle activation levels, posture, and stiffness at phonation onset. However, little work has been ...done investigating the stability of the phonation process in regards to spontaneous changes in vibratory mode of vocal fold oscillation as a function of neuromuscular activation. We evaluated 320 phonatory conditions representing combinations of superior and recurrent laryngeal nerve (SLN and RLN) activations in an in vivo canine model of phonation. At each combination of neuromuscular input, airflow was increased linearly to reach phonation onset and beyond from 300 to 1400 mL/s. High-speed video and acoustic data were recorded during phonation, and spectrograms and glottal-area-based parameters were calculated. Vibratory mode changes were detected based on sudden increases or drops of local fundamental frequency. Mode changes occurred only when SLNs were concurrently stimulated and were more frequent for higher, less asymmetric RLN stimulation. A slight increase in amplitude and cycle length perturbation usually preceded the changes in the vibratory mode. However, no inherent differences between signals with mode changes and signals without were found.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In laryngeal research, studying the vertical vocal fold oscillation component is often disregarded. However, vocal fold oscillation by its nature is a three-dimensional process. In the past, we have ...developed an in-vivo experimental protocol to reconstruct the full, three-dimensional vocal fold vibration. The goal of this study is to validate this 3D reconstruction method. We present an in-vivo canine hemilarynx setup using high-speed video recording and a right-angle prism for 3D reconstruction of vocal fold medial surface vibrations. The 3D surface is reconstructed from the split image provided by the prism. For validation, reconstruction error was calculated for objects located at a distance of up to 15 mm away from the prism. The influence of camera angle, changing calibrated volume, and calibration errors were determined. Overall average 3D reconstruction error is low and does not exceed 0.12 mm at 5 mm distance from the prism. Influence of a moderate (5°) and large (10°) deviation in camera angle led to a slight increase in error to 0.16 mm and 0.17 mm, respectively. This procedure is robust towards changes in calibration volume and small calibration errors. This makes this 3D reconstruction approach a useful tool for the reconstruction of accessible and moving tissue surfaces.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging ...CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 μg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.
We have developed a cell-based outer vocal fold replacement (COVR) as a potential therapy to improve voice quality after vocal fold (VF) injury, radiation, or tumor resection. The COVR consists of ...multipotent human adipose-derived stem cells (hASC) embedded within a three-dimensional fibrin scaffold that resembles vocal fold epithelium and lamina propria layers. Previous work has shown improved wound healing in rabbit studies. In this pilot study in pigs, we sought to develop methods for large animal implantation and phonatory assessment. Feasibility, safety, and structural and functional outcomes of the COVR implant are described. Of eight pigs studied, six animals underwent COVR implantation with harvest between 2 weeks and 6 months. Recovery of laryngeal tissue structure was assessed by vibratory and histologic analyses. Recovery of voice function was assessed by investigating acoustic parameters that were derived specifically for pigs. Results showed improved lamina propria qualities relative to an injured control animal at 6 months. Acoustic parameters reflected voice worsening immediately after surgery as expected; acoustics displayed clear voice recovery in the animal followed for 6 months after COVR. These methods form the basis for a larger-scale long-term pre-clinical safety and efficacy study.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Expression of the disialoganglioside GD2 has been identified as a marker antigen associated with a breast cancer stem-like cell (BCSC) phenotype. Here, we report on the evaluation of GD2 as a ...BCSC-specific target antigen for immunotherapy. GD2 expression was confirmed at variable degree in a set of breast cancer cell lines, predominantly in triple-negative breast cancer (TNBC). To target GD2, we have generated novel anti-GD2 chimeric antigen receptors (GD2-CAR), based on single-chain variable fragments (scFv) derived from the monoclonal antibody (mAb) ch14.18, also known as dinutuximab beta. Expressed on T cells, GD2-CARs mediated specific GD2-dependent T-cell activation and target cell lysis. In contrast to previously described GD2-CARs, no signs of exhaustion by tonic signaling were found. Importantly, application of GD2-CAR expressing T cells (GD2-CAR-T) in an orthotopic xenograft model of TNBC (MDA-MB-231) halted local tumor progression and completely prevented lung metastasis formation. In line with the BCSC model, GD2 expression was only found in a subpopulation (4-6%) of MDA-MB-231 cells before injection. Significant expansion of GD2-CAR-T in tumor-bearing mice as well as T-cell infiltrates in the primary tumor and the lungs were found, indicating site-specific activation of GD2-CAR-T. Our data strongly support previous findings of GD2 as a BCSC-associated antigen. GD2-targeted immunotherapies have been extensively studied in human. In conclusion, GD2-CAR-T should be considered a promising novel approach for GD2-positive breast cancer, especially to eliminate disseminated tumor cells and prevent metastasis formation.
Neutrophils are the first responders in wound healing after injury that mediate pro- and anti-inflammatory activities i.a. through the formation of extracellular traps (NETs). However, excessive NETs ...presence in wound tissue can cause local hyperinflammation and -coagulation resulting in delayed wound healing. To improve wound healing, we aimed to examine the role of NETs and DNase1 on primary and secondary wound healing.
The study included 93 C57BL/6 mice, with 3 different genotypes: wildtype, Pad4-, and DNase1-Knockout (KO). Pad4-KO mice show limited NETs formation, while DNase1-KO mice cannot disintegrate them. All 3 genotypes were included in (1) a laparotomy group and (2) a thermal injury group. Animals in both groups either received DNase1 or a vehicle i.p. post wound induction and wound assessment and euthanasia were conducted. Laparotomy and burn scars were assessed using the stony brook scar evaluation scale and modified Yeong scale respectively. Tissue was analyzed histologically using H&E staining. Ly6g, Collagen I and III, SMA, and Fibrinogen were visualized and neutrophils activation (NE, MPO) and NETs (H3cit) formation assessed.
All animals survived with no complications. DNase1 treatment led to a significantly improved scar appearance in both groups, which was also seen in Pad4-KO mice. In the laparotomy group DNase1 improved collagen deposition and fibrin concentration was significantly reduced by DNase1 treatment. Markers of neutrophil activation were significantly reduced in the treatment and Pad4-KO group. In the thermal injury group wound closure time was significantly reduced after DNase1 treatment and in the Pad4-KO group. Even though inflammation remained high in the thermal injury model over time, neutrophil activation and NETs formation were significantly reduced by DNase1 treatment compared to controls.
Primary and secondary intention wound healing is improved by targeting NETs through DNase1 treatment or genetic KO, as assessed by wound closure time and scar appearances. Additionally, wound stability was not affected by DNASE treatment. The results suggest that overall wound healing is accelerated and DNase1 appears to be a promising option to reduce scar formation; which should be evaluated in humans.
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