CD7 targeted "off-the-shelf" CAR-T demonstrates robust in vivo expansion and high efficacy in the treatment of patients with relapsed and refractory T cell malignancies.

T-cell acute lymphoblastic leukemia (T-ALL) represents an area of highly unmet medical needs. Once relapsed, patients have limited treatment options and poor prognosis. T-ALL antigens such as CD7 is extensively expressed in normal T cells and natural killer (NK) cells, and extending the success of CAR-T therapy to T cell malignancies was challenged by CAR-T cell fratricide, high production cost, and potential product contaminations. GC027 is an "off-the-shelf" allogeneic CD7 targeted CAR-T therapeutic product for T cell malignancies. It demonstrated superior cell expansion and antileukemia efficacy in mouse xenograft model. In our previous study, we observed promising efficacy results in the first two relapsed and refractory(R/R) T-ALL patients treated with GC027. In the expanded study, 11 out of 12 patients had rapid eradication of T-lymphoblasts and reached complete response within 1-month after GC027 infusion. GC027 cells expanded quickly beginning at infusion and reached to peak around 5-10 days after infusion. For most patients with a response(9/11), GC027 could not be detected via flow cytometry or qPCR 4 weeks after infusion. One patient had progression free survival of >3 years. With manageable toxicity profile, GC027 demonstrated superior clinical efficacy to standard chemotherapy regimens in (R/R) T cell malignancies.

Next-day manufacture of a novel anti-CD19 CAR-T therapy for B-cell acute lymphoblastic leukemia: first-in-human clinical study.

To improve clinical outcomes and shorten the vein-to-vein time of chimeric antigen receptor T (CAR-T) cells, we developed the FasT CAR-T (F-CAR-T) next-day manufacturing platform. We report the preclinical and first-in-human clinical studies evaluating the safety, feasibility, and preliminary efficacy of CD19 F-CAR-T in B-cell acute lymphoblastic leukemia (B-ALL). CD19 F-CAR-T cells demonstrated excellent proliferation with a younger cellular phenotype, less exhaustion, and more effective tumor elimination compared to conventional CAR-T cells in the preclinical study. In our phase I study (NCT03825718), F-CAR-T cells were successfully manufactured and infused in all of the 25 enrolled pediatric and adult patients with B-ALL. CD19 F-CAR-T safety profile was manageable with 24% grade 3 cytokine release syndrome (CRS) and 28% grade 3/4 neurotoxicity occurring predominantly in pediatric patients. On day 14, 23/25 patients achieved minimal residual disease (MRD)-negative complete remission (CR), and 20 subsequently underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) within 3 months post F-CAR-T therapy. Fifteen of 20 patients were disease-free with a median remission duration of 734 days. One patient relapsed and 4/20 died from transplant-related mortality. Of the three patients who did not undergo allo-HSCT, two remained in CR until 10 months post-F-CAR-T. Our data indicate that anti-CD19 FasT CAR-T shows promising early efficacy for B-ALL. Further evaluations in larger clinical studies are needed.

Novel CD19 chimeric antigen receptor T cells manufactured next-day for acute lymphoblastic leukemia.

Chimeric antigen receptor-engineered T (CAR-T) cells have shown promising efficacy in patients with relapsed/refractory B cell acute lymphoblastic leukemia (R/R B-ALL). However, challenges remain including long manufacturing processes that need to be overcome. We presented the CD19-targeting CAR-T cell product GC007F manufactured next-day (FasTCAR-T cells) and administered to patients with R/R B-ALL. A total of 21 patients over 14 years of age with CD19+ R/R B-ALL were screened, enrolled and infused with a single infusion of GC007F CAR-T at three different dose levels. The primary objective of the study was to assess safety, secondary objectives included pharmacokinetics of GC007F cells in patients with R/R B-ALL and preliminary efficacy. We were able to demonstrate in preclinical studies that GC007F cells exhibited better proliferation and tumor killing than conventional CAR-T (C-CAR-T) cells. In this investigator-initiated study all 18 efficacy-evaluable patients achieved a complete remission (CR) (18/18, 100.00%) by day 28, with 17 of the patients (94.4%) achieving CR with minimal residual disease (MRD) negative. Fifteen (83.3%) remained disease free at the 3-month assessment, 14 patients (77.8%) maintaining MRD negative at month 3. Among all 21 enrolled patients, the median peak of CAR-T cell was on day 10, with a median peak copy number of 104899.5/µg DNA and a median persistence period of 56 days (range: 7-327 days). The incidence of cytokine release syndrome (CRS) was 95.2% (n = 20), with severe CRS occurring in 52.4% (n = 11) of the patients. Six patients (28.6%) developed neurotoxicity of any grade. GC007F demonstrated superior expansion capacity and a less exhausted phenotype as compared to (C-CAR-T) cells. Moreover, this first-in-human clinical study showed that the novel, next-day manufacturing FasTCAR-T cells was feasible with a manageable toxicity profile in patients with R/R B-ALL.

Social Force Model-Based Safety Evaluation of Intersections in Arterials Considering the Pedestrian Yield Rule.

To enhance the safety of pedestrians crossing the street, a series of new regulations regarding pedestrian yield has been proposed and widely implemented across cities. In this study, we first made some improvements to the social force model, in which pedestrian crossing at the intersection, drivers' psychology of giving way, vehicle yield to pedestrians, vehicle yield in different directions, the influence of pedestrians crossing boundaries, and signal lamp groups on pedestrian behavior were considered. Furthermore, pedestrian crossing and vehicle yield safety models were established, based on which the comprehensive safety evaluation model of intersections in arterials was established, in which two indices-(1) the safety degree of pedestrian crossings and (2) vehicle acceleration interference-were combined with the entropy weight method. Finally, four types of intersections in arterials were studied using a simulation: the intersections between different levels of arterials, and intersections with one-time and two-times pedestrian crossings. Moreover, safety evaluation and analysis of those intersections, considering the rule of pedestrian yield, were conducted combined with the trajectory data from the VISSIM simulation. The relevant results showed that for pedestrians crossing the street, the pedestrian safety of two-time crossing is significantly higher than that of one-time crossing, and compared with the arterial, the pedestrian crossing distance of the sub-arterial is shorter, and the pedestrian perception is safer. Moreover, due to the herd psychology effect, the increase in pedestrian flow volume improves the safety perception of pedestrians at the intersection.

Phase I study of CAR-T cells with PD-1 and TCR disruption in mesothelin-positive solid tumors.

Programmed cell death protein-1 (PD-1)-mediated immunosuppression has been proposed to contribute to the limited clinical efficacy of chimeric antigen receptor T (CAR-T) cells in solid tumors. We generated PD-1 and T cell receptor (TCR) deficient mesothelin-specific CAR-T (MPTK-CAR-T) cells using CRISPR-Cas9 technology and evaluated them in a dose-escalation study. A total of 15 patients received one or more infusions of MPTK-CAR-T cells without prior lymphodepletion. No dose-limiting toxicity or unexpected adverse events were observed in any of the 15 patients. The best overall response was stable disease (2/15 patients). Circulating MPTK-CAR-T cells peaked at days 7-14 and became undetectable beyond 1 month. TCR-positive CAR-T cells rather than TCR-negative CAR-T cells were predominantly detected in effusion or peripheral blood from three patients after infusion. We further confirmed the reduced persistence of TCR-deficient CAR-T cells in animal models. Our results establish the preliminary feasibility and safety of CRISPR-engineered CAR-T cells with PD-1 disruption and suggest that the natural TCR plays an important role in the persistence of CAR-T cells when treating solid tumors.

Ghrelin inhibits IKKß/NF-κB activation and reduces pro-inflammatory cytokine production in pancreatic acinar AR42J cells treated with cerulein.

BACKGROUND: Previous studies have provided conflicting results regarding whether the serum ghrelin concentration can reflect the severity of acute pancreatitis (AP). The present study examined the correlation between the serum ghrelin concentration and AP severity in animal models and investigated whether altered ghrelin expression in pancreatic acinar cells influences IKKß/NF-κB signaling and pro-inflammatory cytokine production. METHODS: Mild or severe AP was induced in rats by intraperitoneal injection of cerulein or retrograde cholangiopancreatic duct injection of sodium taurocholate, respectively. After successful model induction, serum ghrelin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were determined by enzyme-linked immunosorbent assay, and IKKß/NF-κB activation was assessed by immunohistochemistry. Subsequently, stable overexpression or knockdown of ghrelin in AR42J cells was achieved by lentiviral transfection. After transfected cells and control cells were treated with cerulein for 24 h, the TNF-α and IL-1ß levels in the supernatants were determined by enzyme-linked immunosorbent assay, and the expression levels of p-p65, IKKß, and p-IKKß were detected by Western blotting. RESULTS: In rat AP models, AP severity was correlated with increased IKKß/NF-κB activation, pro-inflammatory cytokine production, and ghrelin secretion. The levels of pro-inflammatory cytokines TNF-α and IL-1ß as well as IKKß/NF-κB signaling activity were increased upon knockdown of ghrelin in the AP acinar cell model and decreased with ghrelin overexpression. CONCLUSIONS: Serum ghrelin is related to the severity of AP. Ghrelin may play a protective role in the pathogenesis of AP by inhibiting the pro-inflammatory cytokines and the activation of the IKKß/NF-κB signaling pathway.

Eradication of T-ALL Cells by CD7-targeted Universal CAR-T Cells and Initial Test of Ruxolitinib-based CRS Management.

PURPOSE: Although chimeric antigen receptor T-cell (CAR-T) therapy development for B-cell malignancies has made significant progress in the last decade, broadening the success to treating T-cell acute lymphoblastic leukemia (T-ALL) has been limited. We conducted two clinical trials to verify the safety and efficacy of GC027, an "off-the-shelf" allogeneic CAR-T product targeting T-cell antigen, CD7. Here, we report 2 patients as case reports with relapsed/refractory T-ALL who were treated with GC027. PATIENTS AND METHODS: Both the trials reported here were open-label and single-arm. A single infusion of GC027 was given to each patient after preconditioning therapy. RESULT: Robust expansion of CAR-T cells along with rapid eradication of CD7+ T lymphoblasts were observed in the peripheral blood, bone marrow, and cerebrospinal fluid. Both patients achieved complete remission with no detectable minimal residual disease. At data cutoff, 30 September 2020, 1 of the 2 patients remains in ongoing remission for over 1 year after CAR T-cell infusion. Grade 3 cytokine release syndrome (CRS) occurred in both patients and was managed by a novel approach with a ruxolitinib-based CRS management. Ruxolitinib showed promising activity in a preclinical study conducted at our center. No graft-versus-host disease was observed. CONCLUSIONS: The two case reports demonstrate that a standalone therapy with this novel CD7-targeted "off-the-shelf" allogeneic CAR-T therapy may provide deep and durable responses in select patients with relapsed/refractory T-ALL. GC027 might have a potential to be a promising new approach for treating refractory/relapsed T-ALL. Further studies are warranted.

RF Thermal Plasma Synthesis of Ultrafine ZrB2-ZrC Composite Powders.

Ultrafine ZrB2-ZrC composite powders were synthesized via a radiofrequency (RF) thermal plasma process. Numerical simulation and thermodynamic analysis were conducted to predict the synthesis process, and experimental work was performed accordingly to demonstrate its feasibility. The as-prepared samples were characterized by XRD, FESEM, particle size analyzer, nitrogen/oxygen analyzer, Hall flowmeter, and the Brunner-Emmet-Teller (BET) measurements. The thermodynamic analysis indicated that ZrB2 was preferentially generated, rather than ZrC, and numerical simulation revealed that the solid raw materials could disperse well in the gaseous reactants, and experimental work showed that free carbon particles were easily removed from the products and the elements of Zr, B, C, and O exhibited a uniform distribution. Finally, ZrB2-ZrC composite powders with a particle size of about 100 nm were obtained, the surface area of which was 32.15 m2/g and the apparent density was 0.57 g/cm3.

ß-Galactosidase gene codon optimization results in post-transcriptional enhancement of expression.

OBJECTIVE: lacZ encodes for ß-galactosidase within the galactose operon of bacterial cells. When used as a reporter gene, bacterial "ß-galactosidase" expression is often insufficient for detection in mammalian cells. We intended to optimize the lacZ codon usage according to the most frequently used codons for the seven major proteins in cow's milk, in order to pave a way for the enhancement of transgenic genes expression in eukaryotes. RESULTS: We constructed modified lacZ (named olacZ) according to optional codons used for proteins expressed in cow's milk. The expression of lacZ and olacZ was then compared in HC11 (a murine mammary gland epithelial line), 293T, HeLa, Cos7, and NIH 3T3 cells. While there was no significant difference at the mRNA level between lacZ and olacZ (P > 0.05). The quantification of ß-galactosidase activity and in situ staining experiments showed a 1.2-fold to 3.3-fold expression improvement when comparing olacZ with lacZ. The levels of ß-galactosidase expression at the protein levels from olacZ were approximately 9.2-fold and 2.4-fold respectively for Cos7 and HC11 cells. Furthermore, a 1.9-fold tendency of enhanced expression of olacZ in mammary gland during lactation was observed in transgenic-olacZ mice. CONCLUSION: This study demonstrates an alternative choice for improving lacZ reporter expression in eukaryotes, especially in the mammary gland of cattle or goats.

Multiscale Buffering Engineering in Silicon-Carbon Anode for Ultrastable Li-Ion Storage.

Silicon-carbon (Si-C) hybrids have been proven to be the most promising anodes for the next-generation lithium-ion batteries (LIBs) due to their superior theoretical capacity (∼4200 mAh g-1). However, it is still a critical challenge to apply this material for commercial LIB anodes because of the large volume expansion of Si, unstable solid-state interphase (SEI) layers, and huge internal stresses upon lithiation/delithiation. Here, we propose an engineering concept of multiscale buffering, taking advantage of a nanosized Si-C nanowire architecture through fabricating specific microsized wool-ball frameworks to solve all the above-mentioned problems. These wool-ball-like frameworks, prepared at high yields, nearly matching industrial scales (they can be routinely produced at a rate of ∼300 g/h), are composed of Si/C nanowire building blocks. As anodes, the Si-C wool-ball frameworks show ultrastable Li+ storage (2000 mAh g-1 for 1000 cycles), high initial Coulombic efficiency of ∼90%, and volumetric capacity of 1338 mAh cm-3. In situ TEM proves that the multiscale buffering design enables a small volume variation, only ∼19.5%, reduces the inner stresses, and creates a very thin SEI. The perfect multiscale elastic buffering makes this material more stable compared to common Si nanoparticle-assembled counterpart electrodes.

Modeling and Selection of RF Thermal Plasma Hot-Wall Torch for Large-Scale Production of Nanopowders.

Fouling is a great problem that significantly affects the continuous operation for large-scale radio-frequency (RF) thermal plasma synthesizing nanopowders. In order to eliminate or weaken the phenomenon, numerical simulations based on FLUENT software were founded to investigate the effect of operation parameters, including feeding style of central gas and sheath gas, on plasma torches. It is shown that the tangential feeding style of central gas brings serious negative axial velocity regions, which always forces the synthesized nanopowders to "back-mix", and further leads to the fouling of the quartz tube. Moreover, it is shown that sheath gas should be tangentially fed into the plasma reactor to further eliminate the gas stream's back-mixing. However, when this feeding style is applied, although the negative axial velocity region is decreased, the plasma gas and kinetic energy of the vapor phase near the wall of the plasma reactor are less and lower, respectively; as a result, that plasma flame is more difficult to be arced. A new plasma arcing method by way of feeding gun instead of torch wall was proposed and put in use. The fouling problem has been well solved and plasma arcing is well ensured, and as a result, the experiment on large-scale production of nanopowders can be carried out for 8 h without any interruption, and synthesized Si and Al2O3 nanopowders exhibit good dispersion and sphericity.

Improved biosafety of a lentiviral vector by reducing cellular gene activation.

BACKGROUND: Lentiviral vectors (LVs) have enhancer activity and/or transcriptional read-through (EATRT) properties that can lead to the activation of adjacent genes. Consequently, patients may be at increased risk for adverse effects if such vectors are used clinically. METHODS: In the present study, we assessed the abilities of different "pro-LV"-like constructs with respect to decreasing its EATRT, including the "pro-LV" vector bearing a chimeric ΔLTR of the human foamy virus R-U5 region replaced by that of an LV (HF). RESULTS: By analyzing the EATRT of "pro-LV" constructs transfected in 293T cells, we observed that the inclusion of the first 400 bp of the chicken ß-globin locus HS4 insulator core sequence oriented in the reverse direction (C-) combined with two copies of the simian virus 40 upstream-sequence element (U) at the ΔU3 of ΔLTR region of "pro-LV" tended to shield the adjacent genomic sequences, such that the EATRT rate was lower than when either of the C- or U was included in the "pro-LV". Moreover, upon transduction, the pro-HF appears to reduce the EATRT rate in the chromosomes of 293T (by 80%) and human peripheral blood mononuclear cells (PBMCs) (by 75%) compared to when pro-LV C-U was included (with a 60% and 89% reduction in 293T and PBMCs, respectively). The HF construct had a significant reduction of viral biological titer compared tiowhen the pro-LV C-U was used in 293T cells. CONCLUSIONS: The results of the present study provide an important basis for the clinical applicability of LVs in gene and cell therapy.

Cav 1.2 and Cav 2.2 expression is regulated by different endogenous ghrelin levels in pancreatic acinar cells during acute pancreatitis.

Ghrelin influences pancreatic endocrine and exocrine functions, regulates intracellular calcium [Ca2+]i levels, and has an anti-inflammatory role in acute pancreatitis. This study investigated the role of endogenous ghrelin in the expression of Cav 1.2 (L-type of Ca2+ channel) and Cav 2.2 (N-type of Ca2+ channel) in acute pancreatitis. For this purpose, acute edematous pancreatitis (AEP) and acute necrotizing pancreatitis (ANP) rat models were established. Cav 1.2 and Cav 2.2 expression was assessed by immunohistochemistry in the pancreatic tissues of rats; ghrelin, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) serum levels were detected using ELISA. Next, in AR42J cells with either knock-out or overexpression of ghrelin, Cav 1.2 and Cav 2.2 expression was examined using western blot analysis, and intracellular calcium [Ca2+]i was detected with confocal microscopy. In this study, the ghrelin serum level was highest in the ANP group and was higher in the AEP group than the normal group. Expression of Cav 1.2 and Cav 2.2 in the ANP and AEP groups was higher than in the respective control groups. The serum IL-1ß and TNF-α levels were significantly higher in the ANP group compared to the other groups. Cav 1.2 and Cav 2.2 expression and [Ca2+]i decreased in ghrelin knockdown AR42J cells but increased in ghrelin overexpressing cells. In conclusion, Cav 1.2 and Cav 2.2 expression increased in ANP. The [Ca2+]i level, which is mediated by Cav 1.2 and Cav 2.2 expression, is directly regulated by ghrelin in pancreatic acinar cells, and serum ghrelin levels may be involved in the severity of acute pancreatitis.

Efficacy and Persistence of Allogeneic Adipose-Derived Mesenchymal Stem Cells Combined with Hyaluronic Acid in Osteoarthritis After Intra-articular Injection in a Sheep Model.

Although a number of studies have reported efficacy of autologous adipose-derived mesenchymal stem cells (AD-MSCs) in treating osteoarthritis (OA) no reliable evidences demonstrate whether allogeneic AD-MSCs can efficiently block OA progression in a large animal model. This study explored the efficacy and survival of allogeneic AD-MSCs combined with hyaluronic acid (HA) after intra-articular (IA) injection in a sheep OA model, which were conventionally established by anterior cruciate ligament resection and medial meniscectomy. Allogeneic AD-MSCs from donor sheep at high (5 × 107 cells) and low (1 × 107 cells) doses combined with HA, HA alone, or saline alone were injected into the OA sheep at 3 and 6 weeks after surgery, respectively. Evaluations by magnetic resonance imaging (MRI), macroscopy, micro-computed tomography, and cartilage-specific staining demonstrated that AD-MSCs+HA treated groups preserved typical articular cartilage feature. Inflammatory factors from synovial fluid of AD-MSCs+HA treated groups were significantly lower than those in the HA alone group. Notably, transforming growth factor beta 1 and insulin-like growth factor 1 were detected in the supernatant of cultured AD-MSCs. In addition, labeling signals of allogeneic AD-MSCs could be detected by MRI after 14 weeks of injection and be found in synovium by histology. These results indicated that IA injection of allogeneic AD-MSCs combined with HA could efficiently block OA progression and promote cartilage regeneration and allogeneic AD-MSCs might survive at least 14 weeks after IA injection.

Human adipose-derived mesenchymal progenitor cells engraft into rabbit articular cartilage.

Mesenchymal stem cells (MSCs) are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA). Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs) promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT) and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA) or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE) and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I) but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.

[Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate].

As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of "proviral vectors" in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of LV.

[Progress in improvement of lentiviral vectors' transcriptional read-through].

Four out of 10 patients of X-linked severe combined immunodeficiency (X-SCID) were finally developed leukemia after receiving the treatment of gene therapy delivered by gamma-retroviral vectors. This is due to the vector integrated to the proximity of lmo2 etc proto-oncogene promoters, leading to the activation of onco-gene expression, which raises the concern of the bio-safety of gene therapy vectors. Lentiviral vectors, especially self-inactivating lentiviral vectors, are considered to be much safer than gamma-retroviral vectors. However self-inactivating lentiviral vectors also have encountered with some unsafe factors and one of them is the problem of transcriptional "read-through" . During the past years, achievements have been made to reduce lentiviral vector transcriptional read-through, which are reviewed herein.