Innate-like Gene Expression of Lung-Resident Memory CD8+ T Cells during Experimental Human Influenza: A Clinical Study.

Rationale: Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection. Objectives: To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8+ resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection in vivo. Methods: Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens. Measurements and Main Results: After challenge, 57% of participants became infected. Preexisting influenza-specific CD8+ T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8+ T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8+ T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8+ T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (Cxcl1, Cxcl10, and Cxcl16) that were also expressed by CD8+ cells in bronchial tissues. Conclusions: CD8+ Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).

Genome-edited, donor-derived allogeneic anti-CD19 chimeric antigen receptor T cells in paediatric and adult B-cell acute lymphoblastic leukaemia: results of two phase 1 studies.

BACKGROUND: Genome-edited donor-derived allogeneic anti-CD19 chimeric antigen receptor (CAR) T cells offer a novel form of CAR-T-cell product that is available for immediate clinical use, thereby broadening access and applicability. UCART19 is one such product investigated in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. Two multicentre phase 1 studies aimed to investigate the feasibility, safety, and antileukaemic activity of UCART19 in children and adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. METHODS: We enrolled paediatric or adult patients in two ongoing, multicentre, phase 1 clinical trials to evaluate the safety and antileukaemic activity of UCART19. All patients underwent lymphodepletion with fludarabine and cyclophosphamide with or without alemtuzumab, then children received UCART19 at 1·1-2·3 × 106 cells per kg and adults received UCART19 doses of 6 × 106 cells, 6-8 × 107 cells, or 1·8-2·4 × 108 cells in a dose-escalation study. The primary outcome measure was adverse events in the period between first infusion and data cutoff. These studies were registered at ClinicalTrials.gov, NCT02808442 and NCT02746952. FINDINGS: Between June 3, 2016, and Oct 23, 2018, seven children and 14 adults were enrolled in the two studies and received UCART19. Cytokine release syndrome was the most common adverse event and was observed in 19 patients (91%); three (14%) had grade 3-4 cytokine release syndrome. Other adverse events were grade 1 or 2 neurotoxicity in eight patients (38%), grade 1 acute skin graft-versus-host disease in two patients (10%), and grade 4 prolonged cytopenia in six patients (32%). Two treatment-related deaths occurred; one caused by neutropenic sepsis in a patient with concurrent cytokine release syndrome and one from pulmonary haemorrhage in a patient with persistent cytopenia. 14 (67%) of 21 patients had a complete response or complete response with incomplete haematological recovery 28 days after infusion. Patients not receiving alemtuzumab (n=4) showed no UCART19 expansion or antileukaemic activity. The median duration of response was 4·1 months with ten (71%) of 14 responders proceeding to a subsequent allogeneic stem-cell transplant. Progression-free survival at 6 months was 27%, and overall survival was 55%. INTERPRETATION: These two studies show, for the first time, the feasibility of using allogeneic, genome-edited CAR T cells to treat patients with aggressive leukaemia. UCART19 exhibited in-vivo expansion and antileukaemic activity with a manageable safety profile in heavily pretreated paediatric and adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. The results this study are an encouraging step forward for the field of allogeneic CAR T cells, and UCART19 offers the opportunity to treat patients with rapidly progressive disease and where autologous CAR-T-cell therapy is unavailable. FUNDING: Servier.

How a dynamic optical system maintains image quality: Self-adjustment of the human eye.

The eyeball is continually subjected to forces that cause alterations to its shape and dimensions, as well as to its optical components. Forces that induce accommodation result in an intentional change in focus; others, such as the effect of intraocular pressure fluctuations, are more subtle. Although the mechanical properties of the eyeball and its components permit mediation of such subtle forces, the concomitant optical changes are not detected by the visual system. Optical self-adjustment is postulated as the mechanism that maintains image quality. The purpose of this study was to investigate how self-adjustment occurs by using an optical model of the eyeball and to test the requisite optical and biometric conditions.

Data analysis of the ocular response analyzer for improved distinction and detection of glaucoma.

The purpose of this study was to evaluate the clinical utility of the output parameters of the ocular response analyzer (ORA) and those calculated from the raw ORA in subjects with healthy eyes and those with suspected glaucoma, and in patients with two types of glaucoma. The raw ORA data were analyzed using a custom software that included the Gaussian filtering of applanation curves for three different window sizes. To the best of our knowledge, these findings present a novel means of optimizing the use of measurements from the ORA, which can refine the characteristics of corneal biomechanics, enabling a distinction between the types of glaucoma and leading to an improvement in diagnosing and early detection.

Diagnostic utility of monitoring cytomegalovirus-specific immunity by QuantiFERON-cytomegalovirus assay in kidney transplant recipients.

BACKGROUND: Despite universal prophylaxis, late cytomegalovirus (CMV) infection occurs in a high proportion of kidney transplant recipients. We evaluated whether a specific viral T-cell response allows for the better identification of recipients who are at high risk of CMV infection after prophylaxis withdrawal. METHODS: We conducted a prospective study in 19 pretransplant anti-CMV seronegative kidney graft recipients R- (18 from seropositive donors [D+] and one from a seronegative donor [D-]) and 67 seropositive recipients R(+) (59 from seropositive donors and eight from seronegative donors) who received antiviral prophylaxis with valganciclovir. The QuantiFERON-CMV (QF-CMV) assay was performed within the first and third months after transplantation. Blood samples were monitored for CMV DNAemia using a commercial quantitative nucleic acid amplification test (QNAT) that was calibrated to the World Health Organization International Standard. RESULTS: Twenty-one of the 86 patients (24%) developed CMV viremia after prophylaxis withdrawal within 12 months posttransplantation. In the CMV R(+) group, the QF-CMV assay yielded reactive results (QF-CMV[+]) in 51 of 67 patients (76%) compared with 7 of 19 patients (37%) in the CMV R(-) group (p = 0.001). In the CMV R(+) group, infection occurred in seven of 16 recipients (44%) who were QF-CMV(-) and eight of 51 recipients (16%) who were QF-CMV(+). In the CMV R(-) group, infection evolved in five of 12 recipients (42%) who were QF-CMV(-) and one of 7 recipients (14%) who were QF-CMV(+). No difference was found in the incidence of CMV infection stratified according to the QF-CMV results with regard to the recipients' pretransplant CMV IgG serology (p = 0.985). Cytomegalovirus infection occurred in 15 of 36 patients (42%) with hypogammaglobulinemia (HGG) 90 days posttransplantation compared with two of 34 patients (6%) without HGG (p = 0.0004). Cytomegalovirus infection occurred in seven of 13 patients (54%) with lymphocytopenia compared with 14 of 70 patients (20%) without lymphocytopenia (p = 0.015). The multivariate analysis revealed that the nonreactive QuantiFERON-CMV assay was an independent risk factor for postprophylaxis CMV infection. CONCLUSIONS: In kidney transplant recipients who received posttransplantation prophylaxis, negative QF-CMV results better defined the risk of CMV infection than initial CMV IgG status after prophylaxis withdrawal. Hypogammaglobulinemia and lymphocytopenia were risk factors for CMV infection.

Cathelicidins Have Direct Antiviral Activity against Respiratory Syncytial Virus In Vitro and Protective Function In Vivo in Mice and Humans.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection.

Eye retraction and rotation during Corvis ST 'air puff' intraocular pressure measurement and its quantitative analysis.

PURPOSE: The aim of this study was to analyse the indentation and deformation of the corneal surface, as well as eye retraction, which occur during air puff intraocular pressure (IOP) measurement. METHODS: A group of 10 subjects was examined using a non-contact Corvis ST tonometer, which records image sequences of corneas deformed by an air puff. Obtained images were processed numerically in order to extract information about corneal deformation, indentation and eyeball retraction. RESULTS: The time dependency of the apex deformation/eye retraction ratio and the curve of dependency between apex indentation and eye retraction take characteristic shapes for individual subjects. It was noticed that the eye globes tend to rotate towards the nose in response to the air blast during measurement. This means that the eye globe not only displaces but also rotates during retraction. Some new parameters describing the shape of this curve are introduced. Our data show that intraocular pressure and amplitude of corneal indentation are inversely related (r8  = -0.83, P = 0.0029), but the correlation between intraocular pressure and amplitude of eye retraction is low and not significant (r8  = -0.24, P = 0.51). CONCLUSIONS: The curves describing corneal behaviour during air puff tonometry were determined and show that the eye globe rotates towards the nose during measurement. In addition, eye retraction amplitudes may be related to elastic or viscoelastic properties of deeper structures in the eye or behind the eye and this should be further investigated. Many of the proposed new parameters present comparable or even higher repeatability than the standard parameters provided by the Corvis ST.

Impaired Antibody-mediated Protection and Defective IgA B-Cell Memory in Experimental Infection of Adults with Respiratory Syncytial Virus.

RATIONALE: Despite relative antigenic stability, respiratory syncytial virus (RSV) reinfects throughout life. After more than 40 years of research, no effective human vaccine exists and correlates of protection remain poorly defined. Most current vaccine candidates seek to induce high levels of RSV-specific serum neutralizing antibodies, which are associated with reduced RSV-related hospitalization rates in observational studies but may not actually prevent infection. OBJECTIVES: To characterize correlates of protection from infection and the generation of RSV-specific humoral memory to promote effective vaccine development. METHODS: We inoculated 61 healthy adults with live RSV and studied protection from infection by serum and mucosal antibody. We analyzed RSV-specific peripheral blood plasmablast and memory B-cell frequencies and antibody longevity. MEASUREMENTS AND MAIN RESULTS: Despite moderately high levels of preexisting serum antibody, 34 (56%) became infected, of whom 23 (68%) developed symptomatic colds. Prior RSV-specific nasal IgA correlated significantly more strongly with protection from polymerase chain reaction-confirmed infection than serum neutralizing antibody. Increases in virus-specific antibody titers were variable and transient in infected subjects but correlated with plasmablasts that peaked around Day 10. During convalescence, only IgG (and no IgA) RSV-specific memory B cells were detectable in peripheral blood. This contrasted with natural influenza infection, in which virus-specific IgA memory B cells were readily recovered. CONCLUSIONS: This observed specific defect in IgA memory may partly explain the ability of RSV to cause recurrent symptomatic infections. If so, vaccines able to induce durable RSV-specific IgA responses may be more protective than those generating systemic antibody alone.

In vivo longitudinal chromatic aberration of pseudophakic eyes.

PURPOSE: To present the results of longitudinal chromatic aberration measurements on two groups of pseudophakic eyes in comparison to healthy eyes. METHODS: The longitudinal chromatic aberration of the eye, defined as chromatic difference of refraction with disabled accommodation, was measured with the use of a visual refractometer with a custom-designed target illuminator consisting of a narrow-band RGB diode (blue λb = 470 ± 15 nm; green λg = 525 ± 18 nm; red λr = 660 ± 10 nm). The measurements were performed on nine eyes implanted with AcrySof IQ SN60WF, 14 eyes implanted with AcrySof SA60AT, and 10 phakic eyes under cycloplegia. RESULTS: The mean values of the longitudinal chromatic aberration between 470 and 660 nm for the control group was 1.12 ± 0.14 D. For SA60AT group, it was 1.45 ± 0.42 D whereas for SN60WF it was 1.17 ± 0.52 D. The statistical test showed significant difference between SA60AT and the control group (p << 0.05) and no significant difference between SN60WF and the control groups (p = 0.64). CONCLUSIONS: The study showed that the longitudinal chromatic aberration in vivo can be easily and reliably estimated with an adapted visual refractometer. The two groups of pseudophakic eyes measured in this study showed different values of chromatic aberration. Its magnitude for SA60AT group was significantly larger than for the control group whereas for SN60WF the difference was not significant. The optical material used for intraocular lens design may have significant influence on the magnitude of the chromatic aberration of the pseudophakic eye, and therefore on its optical and visual performance in polychromatic light.

An integrated model of the human cornea as a linear biaxial birefringent medium.

A novel model of human corneal birefringence is presented. The cornea is treated as a homogeneous biaxial linear birefringent medium in which the values of the binormal axes angle and organization of the main refractive indices vary continuously from the apex to the limbus. In its central part, the angle between binormal axes is 35°, and para centrally, it smoothly increases to 83.7°. The values of the main refractive indices (nx, ny, nz) change, as well as their order, from nx < nz < ny to nz < nx < ny. The transition between these two states was described with a normal distribution (µ = 0.45, σ = 0.1). The presented model corresponds with the experimental results presented in the literature. To our knowledge, it is the first model that presents the anisotropic properties' distributions of the entire cornea. The presented model facilitates a better understanding of the corneal birefringence phenomenon directly related to its lamellar structure.

Investigation of crystalline lens overshooting: ex vivo experiment and optomechanical simulation results.

Introduction: Crystalline lens overshooting refers to a situation in which the lens momentarily shifts too much from its typical location immediately after stopping the rotational movement of the eye globe. This movement can be observed using an optical technique called Purkinje imaging. Methods: In this work, an experimental setup was designed to reproduce this effect ex vivo using a fresh porcine eye. The sample was rotated 90° around its centroid using a high-velocity rotation stage, and the Purkinje image sequences were recorded, allowing us to quantify the overshooting effect. The numerical part of the study consisted of developing a computational model of the eye, based on the finite element method, that allowed us to understand the biomechanical behavior of the different tissues in this dynamic scenario. A 2D fluid-structure interaction model of the porcine eye globe, considering both the solid parts and humors, was created to reproduce the experimental outcomes. Results: Outputs of the simulation were analyzed using an optical simulation software package to assess whether the mechanical model behaves optically like the real ex vivo eye. The simulation predicted the experimental results by carefully adjusting the mechanical properties of the zonular fibers and the damping factor. Conclusion: This study effectively demonstrates the importance of characterizing the dynamic mechanical properties of the eye tissues to properly comprehend and predict the overshooting effect.

Opto-mechanical self-adjustment model of the human eye.

The eye has specific optical and biomechanical properties that jointly regulate the eye's quality of vision, shape, and elasticity. These two characteristics are interdependent and correlated. Contrary to most currently available computational models of the human eye that only focus on biomechanical or optical aspects, the current study explores the inter-relationships between biomechanics, structure, and optical properties. Possible combinations of mechanical properties, boundary conditions, and biometrics were specified to ensure the opto-mechanical (OM) integrity to compensate for physiological changes in intraocular pressure (IOP) without compromising image acuity. This study evaluated the quality of the vision by analyzing the minimum spot diameters formed on the retina and drew how the self-adjustment mechanism affects the eye globe shape by adopting a finite element (FE) model of the eyeball. The model was verified by a water drinking test with biometric measurement (OCT Revo NX, Optopol) and tonometry (Corvis ST, Oculus).

Selected Predictors of COVID-19 Mortality in the Hospitalised Patient Population in a Single-Centre Study in Poland.

Background: The correct analysis of COVID-19 predictors could substantially improve the clinical decision-making process and enable emergency department patients at higher mortality risk to be identified. Methods: We retrospectively explored the relationship between some demographic and clinical factors, such as age and sex, as well as the levels of ten selected factors, namely, CRP, D-dimer, ferritin, LDH, RDW-CV, RDW-SD, procalcitonin, blood oxygen saturation, lymphocytes, and leukocytes, and COVID-19 mortality risk in 150 adult patients diagnosed with COVID-19 at Provincial Specialist Hospital in Zgierz, Poland (this hospital was transformed, in March 2020, into a hospital admitting COVID-19 cases only). All blood samples for testing were collected in the emergency room before admission. The length of stay in the intensive care unit and length of hospitalisation were also analysed. Results: The only factor that was not significantly related to mortality was the length of stay in the intensive care unit. The odds of dying were significantly lower in males, patients with a longer hospital stay, patients with higher lymphocyte levels, and patients with higher blood oxygen saturation, while the chances of dying were significantly higher in older patients; patients with higher RDW-CV and RDW-SD levels; and patients with higher levels of leukocytes, CRP, ferritin, procalcitonin, LDH, and D-dimers. Conclusions: Six potential predictors of mortality were included in the final model: age, RDW-CV, procalcitonin, and D-dimers level; blood oxygen saturation; and length of hospitalisation. The results obtained from this study suggest that a final predictive model with high accuracy in mortality prediction (over 90%) was successfully built. The suggested model could be used for therapy prioritization.

Complexity of crystalline lens wobbling investigated by means of combined mechanical and optical simulations.

Crystalline lens wobbling is a phenomenon when the lens oscillates briefly from its normal position immediately after stopping the rotational movement of the eye globe. It can be observed by means of Purkinje imaging. The aim of this research is to present the data and computation workflow that involve both biomechanical and optical simulations that can mimic this effect, aimed to better understanding of lens wobbling. The methodology described in the study allows to visualize both the dynamic changes of the lens conformation within the eye and its optical effect in terms of Purkinje performance.

Impact of JNK1, JNK2, and ligase Itch on reactive oxygen species formation and survival of prostate cancer cells treated with diallyl trisulfide.

PURPOSE: In our previous study, we demonstrated that diallyl trisulfide (DATS) induced iron-dependent G2-M arrest of prostate cancer cell cycle. Moreover, ferritin degradation and an increase of labile iron pool has been linked to the activation of the JNK signaling axis. In the present work, we extended this study to determine which of the c-jun kinases is responsible for ferritin degradation and the role of iron in DATS-induced cell death. We hypothesized that JNK1 activates Itch ligase which will lead to ferritin ubiquitination, an increase in iron-dependent ROS formation and cell death. METHODS: PC-3 prostate cancer cells were used in this study. Cell viability, concentration of ROS, labile iron pool, and changes in ferritin and P-Itch and DNA damage were determined. RESULTS: We observed that DATS induced ferritin degradation through JNK, Itch signaling axis. DATS did not induce neither ROS formation nor increase the LIP in JNK1-DN transfected cells. We also observed that DATS increased JNK-dependent activating phosphorylation of E3ligase Itch. The cells transfected with inactive form of Itch were more resistant against cytotoxicity of DATS and showed lower DATS-induced ferritin degradation. Desferrioxamine a specific iron chelator had no effect neither on cell viability nor DNA damage evaluated by comet assay. CONCLUSIONS: These results suggest that JNK1-dependent increase in LIP is mediated by Itch ubiquitin ligase.

Corneal Behavior during Tonometer Measurement during the Water Drinking Test in Eyes with XEN GelStent in Comparison to Non-Implanted Eyes.

Biomechanics of the cornea have significant influences on the non-contact measurement of the intraocular pressure. The corneal behaviour during tonometry is a fundamental factor in estimating its value. The aim of the study was to analyse the behaviour of the cornea during tonometric measurement with the forced change in intraocular pressure during the water drinking test. Ocular Response Analyser (Reichert) was used to the measurement. Besides four basic parameters connected with intraocular pressure (IOPg, IOPcc) and biomechanics (corneal hysteresis CH and corneal resistance factor (CRF), other parameters representing the behaviour of the cornea during a puff of air were analysed. There were 47 eyes included in the study, including 27 eyes with a XEN GelStent implanted and 20 without it. The eyes of people with monocular implementation were the reference group. The values of analysed parameters were compared before and after 10, 25, 40, and 55 min after drinking the water. The intraocular pressure increased by 2.4 mmHg (p < 0.05) for eyes with a XEN stent and 2.2 mmHg for eyes without a stent (p < 0.05) in the tenth minute after drinking of water. This change caused a decreasing of corneal hysteresis (p < 0.05) without significant changes in the corneal resistance factor (p > 0.05). Corneal hysteresis changed similarly in the reference group and the group with a XEN GelStent. The analysis of additional parameters showed a difference in the behaviour of the cornea in eyes with a XEN GelStent in comparison to the corneas of eyes without a stent. This was particularly visible in the analysis of the cornea's behaviour during the second applanation, when the cornea returns to its baseline state after deformation caused by air puff tonometry.

Clinical Pharmacology and Determinants of Response to UCART19, an Allogeneic Anti-CD19 CAR-T Cell Product, in Adult B-cell Acute Lymphoblastic Leukemia.

Background: UCART19 is an "off-the-shelf" genome-edited anti-CD19 chimeric antigen receptor (CAR)-T cell product, manufactured from unrelated healthy donor cells. Methods: UCART19 was administered to 25 adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) in the CALM trial. All patients underwent lymphodepletion with fludarabine and cyclophosphamide ± alemtuzumab and received one of three ascending doses of UCART19. Given the allogeneic nature of UCART19, we analyzed the impact of lymphodepletion, HLA disparities, and host immune system reconstitution on its kinetics, along with other factors known to affect autologous CAR-T cell clinical pharmacology. Results: Responder patients (12/25) had higher UCART19 expansion (C max) and exposure (AUCTlast) than nonresponders (13/25), as measured by transgene levels in peripheral blood. The persistence of CAR+ T cells did not exceed 28 days in 10/25 patients and lasted beyond 42 days in 4/25. No significant correlation was found between UCART19 kinetics and administered cell dose, patient and product characteristics or HLA disparities. However, the number of prior lines of therapy and absence of alemtuzumab negatively impacted UCART19 expansion and persistence. Alemtuzumab exposure positively affected IL7 and UCART19 kinetics, while negatively correlating with host T lymphocyte AUC0-28. Conclusions: UCART19 expansion is a driver of response in adult patients with R/R B-ALL. These results shed light on the factors associated with UCART19 kinetics, which remain highly affected by the impact of alemtuzumab on IL7 and host-versus-graft rejection. Significance: First description of the clinical pharmacology of a genome-edited allogeneic anti-CD19 CAR-T cell product showing the crucial role of an alemtuzumab-based regimen in sustaining UCART19 expansion and persistence through increased IL7 availability and decreased host T lymphocyte population.

Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

UCART19, a first-in-class allogeneic anti-CD19 chimeric antigen receptor T-cell therapy for adults with relapsed or refractory B-cell acute lymphoblastic leukaemia (CALM): a phase 1, dose-escalation trial.

BACKGROUND: The prognosis for adults with relapsed or refractory B-cell acute lymphoblastic leukaemia remains poor. UCART19, an allogeneic genome-edited anti-CD19 chimeric antigen receptor (CAR) T-cell product derived from healthy donors and available for immediate clinical use, offers a potential therapeutic option for such patients. The CALM trial is a first-in-human study evaluating the safety and antileukaemic activity of UCART19 in adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. METHODS: This phase 1, open-label study was conducted at eight centres across France, the UK, the USA, and Japan. Adult patients aged 16-70 years with CD19-positive relapsed or refractory B-cell acute lymphoblastic leukaemia who had morphological relapse or a minimal residual disease level of at least 1 × 10-3 and had exhausted standard treatment options were enrolled in the study, which comprised a dose-escalation phase of up to three UCART19 doses followed by a safety expansion phase. Patients underwent lymphodepletion with fludarabine (30 mg/m2 per day intravenously for 3 days) and cyclophosphamide (500 mg/m2 per day intravenously for 3 days) with or without alemtuzumab (1 mg/kg or 40 mg or 60 mg over 5 days) and received UCART19 doses of 6 × 106, 6-8 × 107, or 1·8-2·4 × 108 total CAR T cells intravenously, followed by safety evaluation and disease response assessments. The primary endpoint was incidence and severity of adverse events. Secondary endpoints were the overall response rate, duration of response, relapse-free survival, progression-free survival, and overall survival. This trial is registered with ClinicalTrials.gov (NCT02746952) and is complete. FINDINGS: Between Aug 1, 2016, and June 30, 2020, 25 patients were enrolled in the study and treated with UCART19. Median duration of follow-up was 12·8 months (IQR 2·8-24·8). Median age was 37 years (IQR 28-45). 14 (56%) patients were male and 11 (44%) female. 17 (68%) patients were White, two (8%) Black, two (8%) Asian, and four (16%) from other racial or ethnic groups. Three patients developed dose-limiting toxicities (one at each dose level); one had grade 4 cytokine release syndrome and two had grade 4 prolonged cytopenias. Grade 3 or higher cytokine release syndrome was reported in six (24%) patients and grade 3 or higher neurological toxicity in one (4%) patient. Grade 3 or higher infections occurred in seven (28%) patients, and grade 4 prolonged cytopenia in four (16%) patients. Two (8%) patients developed grade 1 acute cutaneous graft-versus-host disease. 14 patients died, nine from progressive disease and five from infections or other complications, of which four were considered to be related to UCART19 or lymphodepletion, or both. After a median of follow-up of 12·8 months (IQR 2·8-24·8), overall response rate was 48% (95% CI 28-69; 12 of 25 patients), duration of response and median relapse-free survival were 7·4 months (95% CI 1·8 to not calculable), progression-free survival was 2·1 months (95% CI 1·2-2·8), and overall survival was 13·4 months (95% CI 4·8-23·0). INTERPRETATION: UCART19 had a manageable safety profile, and showed evidence of antileukaemic activity in heavily pretreated adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. This study shows that allogeneic off-the-shelf CAR T cells can be used safely to treat patients with relapsed B-cell acute lymphoblastic leukaemia. FUNDING: Servier.