First-in-Human Trial of EphA2-Redirected CAR T-Cells in Patients With Recurrent Glioblastoma: A Preliminary Report of Three Cases at the Starting Dose

Glioblastoma is the most common primary brain malignancy with limited treatment options. EphA2 is a tumor-associated-antigen overexpressed in glioblastoma. Pre-clinical studies have demonstrated the promise of EphA2-redirected CAR T-cells against glioblastoma. We conduct the first-in-human trial of EphA2-redirected CAR T-cells in patients with EphA2-positive recurrent glioblastoma and report the results of three patients enrolled as the first cohort receiving the starting dosage (1×106 cells/kg). A single infusion of EphA2-redirected CAR T-cells was administrated intravenously, with the lymphodepletion regimen consisting of fludarabine and Cyclophosphamide. In two patients, there was grade 2 cytokine release syndrome accompanied by pulmonary edema, which resolved completely with dexamethasone medication. Except that, there was no other organ toxicity including neurotoxicity. In both the peripheral blood and cerebral-spinal-fluid, we observed the expansion of CAR T-cells which persisted for more than four weeks. In one patient, there was a transit diminishment of the tumor. Among these three patients, one patient reported SD and two patients reported PD, with overall survival ranging from 86 to 181 days. At the tested dose level (1×106 cells/kg), intravenously infusion of EphA2-rediretected CAR T-cells were preliminary tolerable with transient clinical efficacy. Future study with adjusted dose and infusion frequency of CAR T-cells is warranted.Trial Registration NumbersNCT 0342399

Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement

Nanocrystals in compression: unexpected structural phase transition and amorphization due to surface impurities.

We report an unprecedented surface doping-driven anomaly in the compression behaviors of nanocrystals demonstrating that the change of surface chemistry can lead to an interior bulk structure change in nanoparticles. In the synchrotron-based X-ray diffraction experiments, titania nanocrystals with low concentration yttrium dopants at the surface are found to be less compressible than undoped titania nanocrystals. More surprisingly, an unexpected TiO2(ii) phase (α-PbO2 type) is induced and obvious anisotropy is observed in the compression of yttrium-doped TiO2, in sharp contrast to the compression behavior of undoped TiO2. In addition, the undoped brookite nanocrystals remain with the same structure up to 30 GPa, whereas the yttrium-doped brookite amorphizes above 20 GPa. The abnormal structural evolution observed in yttrium-doped TiO2 does not agree with the reported phase stability of nano titania polymorphs, thus suggesting that the physical properties of the interior of nanocrystals can be controlled by the surface, providing an unconventional and new degree of freedom in search for nanocrystals with novel tunable properties that can trigger applications in multiple areas of industry and provoke more related basic science research

ORF5_CY2 forms VLPs in infected <i>N</i>. <i>benthamiana</i>.

(A) CY1- and CY2-infected plants were subjected to ultracentrifugation to gently purify any VLPs. Material from CY1- and CY2-infected N. benthamiana in the 70% sucrose cushion, interface (Int), and 25% sucrose cushion was examined following electrophoresis through a 0.8% agarose gel. Nb, N. benthamiana. (B) Western blot detection of ORF5CY2. Total proteins from healthy N. benthamiana and CY2-infected N. benthamiana, and 70% sucrose fractions of CY1- and CY2-infected N. benthamiana were separated by 10% PAGE. ORF5CY2-specific antibody was used to detect ORF5CY2 (arrow). (C) Northern blot detection of CY2 gRNA and sgRNA in material from the 70% sucrose fraction of CY2-infected N. benthamiana. Total RNA from uninfected N. benthamiana and CY2-infected N. benthamiana served as negative and positive controls, respectively. (D) Samples of 70% sucrose cushions from uninfected N. benthamiana, CY1-infected N. benthamiana, and CY2-infected N. benthamiana were exchanged to PBS buffer and observed by EM. Particles of 3 different sizes were found in CY2-infected N. benthamiana that were absent from uninfected and CY1-infected N. benthamiana. Yellow arrows point to CY2-infected N. benthamiana particles with a diameter of 14 nm that were the most prevalent. Red arrow denotes a 24 nm particle and blue arrow denotes a 35 nm particle. EM, electron microscopy; N b., N. benthamiana; ORF, open reading frame; VLP, virus-like particle.</p

CY2 replicates and moves systemically in the absence of ORF5.

(A) Secondary structures of a portion of CY1 (left) and the corresponding region of CY2 (right). Sequence shaded light green in CY2 is a “carmovirus consensus sequence” (G2-3A/U5-9) located at the 5′ ends of carmovirus and umbravirus gRNAs and sgRNAs. Red residues differ between CY1 and CY2. ORF5 initiation codon is circled and shaded yellow. CY2 mutants CY2sgm1 and CY2sgm2 have color-coded alterations in the sgRNA promoter region. CY25T contains a truncated ORF5 protein (33 amino acids remaining). (B) Accumulation of (+)- and (-)-strands of CY1, CY2, and CY2 mutants in Arabidopsis protoplasts at 24 h and 48 h post-transfection assayed by northern blots. CY1 accumulation was low but detectable. CY1 with an RdRp active site alteration (CY1-GDD) served as a negative control. Mock, no added RNA. Data was obtained from 3 independent experiments. Standard deviation is shown. No significant difference in accumulation of the gRNA was found for CY2 and the CY2 mutants using JMP Pro 16, Student’s t, α = 0.05. (C) Competition assays between CY1 and WT and mutant CY2. Symptomatic systemic leaves were collected at 3-, 4-, and 6-wpi and total RNA was extracted and subjected to northern analysis. CY1 and CY2 size differences were used to visually assess levels of the different gRNAs. Three independent experiments were conducted with very similar results. Note that while WT CY2 is mainly detected at 3-wpi in co-infiltrated plants, both CY1 and CY2 are present at 4- and 6-wpi (left panel). In contrast, CY1 is mainly detected at all time points in plants co-infiltrated with the ORF5 deficient or defective mutants (middle and right panels). ●, denotes low level band that is not detected in CY2sgm1 protoplasts infections that is near the size of the sgRNA (831 nt) and is also the size of a defective (D)-RNA (921 nt) that commonly arises in CY1/CY2 infections. ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; wpi, weeks post-infiltration; WT, wild type.</p

CY2 and CY2sgm1 accumulated in phloem tissues of stem, petiole, and roots.

FISH analysis of CY1, CY2, and CY2sgm1 at 60 dpi. Cy3-labeled oligonucleotide probe targeted CY1 and CY2 positions 1101–1131. DAPI-stained DNA and xylem tissue fluoresce blue. Bar = 50 μm. No viral RNA signals were detected in non-phloem tissue. (PDF)</p

ORF5_CY2 is structurally similar to 30K MPs and luteovirus/polerovirus/sobemovirus CPs.

(A) Structure modeling of full-length ORF5CY2 using Alphafold2. Color coding from blue to red indicates high to low structural confidence, respectively. Unstructured N-terminal region contains a predicted nucleolar localization signal (NoLS). (B) Overlay images of ORF5CY2-GFP and RFP-NoLS in N. benthamiana. Leaves were co-infiltrated with Agrobacteria expressing ORF5CY2-GFP and RFP-NoLS from CaMV 35S promoters. Infiltrated leaves were collected at 2 dpi and subjected to confocal microscopy. (C) Single jelly-roll domains of ORF5CY2, OuMV MP (MPOuMV), and BYDV P3 CP (CPBYDV). Full-length structures of MPOuMV and CPBYDV are boxed. CPBYDV also contains an N-terminal NoLS. (D) Maximum-likelihood phylogenetic tree based on amino acid sequences of Class 2 ORF5 (blue), representative 30K MPs (red), and SeMV (sobemovirus), BYDV (luteovirus), and PLRV (polerovirus) CPs (green). Branch numbers indicate bootstrap support in percentage out of 1,000 replicates. The letters beneath the branch numbers indicate the node names. The scale bar denotes amino acid substitutions per site. The tree is mid-point rooted. Protein sequences were aligned through PROMALS3D and subjected to phylogenetic analysis in MEGA X. BYDV, barley yellow dwarf virus; CP, capsid protein; dpi, days post-infiltration; MP, movement protein; ORF, open reading frame; OuMV, ourmia melon virus; PLRV, potato leafroll virus; SeMV, sesbania mosaic virus.</p