Multiplex measurements of the impacts of ACE2 sequence and cell surface abundance during SARS-like coronavirus entry

SARS-like coronaviruses, including SARS-CoV and SARS-CoV-2, encode spike proteins that bind human ACE2 protein on the cell surface to enter target cells and cause infection. The efficiency of virus entry depends on ACE2 sequence and expression levels in target cells. A small fraction of humans encodes variants of ACE2, thus altering the biochemical properties at the protein interaction interface. All humans possess cells with vastly differing amounts of ACE2 on the cell surface, ranging from cell types with high expression in the gut and lungs to lower expression in the liver and pancreas. Mastering our understanding of spike-ACE2 interaction and infection requires experiments precisely perturbing both variables. Thus, we developed a synthetic cell engineering approach compatible with high throughput assays for pseudo-typed virus infection. Our assay system is capable of assessing both variables individually and in combination. We adapted an engineered HEK293T DNA recombinase landing pad cell line capable of expressing transgenic ACE2 sequences at highly precise levels. Infection with lentiviruses pseudotyped with the spikes of SARS-like coronaviruses revealed that high ACE2 abundance could mask the effects of impaired binding thereby making it challenging to know the role of affinity altering mutations during infection. We limited the ACE2 abundance on the cell surface by expressing transgenic ACE2 behind a suboptimal Kozak sequence, thereby altering its protein translation rate. This allowed us to understand how ACE2 sequence could impact its interaction with coronavirus spike proteins as two human ACE2 variants at the binding interface, K31D and D355N, exhibited reduced infection. Our experiments suggested that we need to better understand how ACE2 expression determines the susceptibility of cells for SARS-like coronavirus binding and infection. We thus created an ACE2 Kozak library consisting of ~4,096 Kozak variants, each conferring a different ACE2 protein translation rate thus resulting in a range of ACE2 steady-state abundances. Combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) revealed the library to span two orders of magnitude of ACE2 abundance. Challenging this library of cells with spike pseudotyped lentiviruses revealed how ACE2 abundance correlated with infection rate. The library-based experiments yielded a dynamic range wider than traditional single sample infection assay, likely more representative of infection dynamics in vivo. Now that we have characterized the impacts of ACE2 abundance on infectivity in engineered cells, our next goal is to expand the comparison to physiologically relevant cells with endogenously expressed proteins. Modulating protein abundance levels will be key to creating maximally informative functional assays for any protein in cell-based assays, and we have laid the groundwork for being able to simultaneously test the impacts of protein abundance and sequence in combination for proteins involved in diverse cellular processes. This research was supported by a National Institute of Health (NIH) grant GM142886 (KAM).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

CD8+ T CELL CLONES FROM PRIOR SARS-CoV-2 INFECTION PREDOMINATE AFTER mRNA VACCINATION

Background: Hybrid immunity is more protective than vaccination or prior infection alone. To understand the formation of hybrid immunity, we studied how SARS-CoV-2 mRNA vaccines interact with T cell memory by tracking spike (S) specific T cells in cohorts of hospitalized (n = 19) or non-hospitalized (n = 34) COVID-19 convalescents. We hypothesized that S-reactive CD4 and CD8 T cells would increase in response to serial vaccine doses and reflect prior immune exposure at the clonal level. Method(s): After vaccination, we stimulated PBMCs from 12 participants (8M/4F) with peptides spanning S. Activated cells (CD69+CD137+) were sorted and CD4/CD8 phenotype linked with paired TRB-TRA sequences at single cell resolution. S-reactive TRB sequences were mapped within 4-6 serial blood and post-booster nasal TRB repertoires to evaluate S-reactive CD4 and CD8 T cell clonotypic kinetics spanning convalescence to boost. PBMCs from 53 participants were sequenced with the ImmunoSEQ assay to evaluate S-reactive TRB breadth using a database of S-assigned TRB sequences (Adaptive Biotechnologies), comparing S-reactive TRB diagnostic breadth by hospitalization status (Wilcoxon test). Result(s): SARS-CoV-2 mRNA vaccination provoked strong T cell clonal expansion in most participants. At 8-12 months after infection, each primary mRNA dose increased the abundance and diversity of S-specific T cells. Clonal and integrated expansions were larger in CD8 than in CD4 T cells. At the convalescent time point, we observed greater diagnostic S-reactive CD4 T cell breadth in hospitalized vs. non-hospitalized patients (p< 0.01). CD4 T cell S breadth was again higher in previously hospitalized persons after the 2nd primary (p=0.02) and booster (p< 0.01) doses, suggesting that diverse CD4 T cell memory after severe infection leads to increased repertoire diversity after vaccination. S-specific T cells with identical TCRs were detectable in blood and the nasal mucosa, with specificity confirmed using a TRA/TRB transgenic T cell with the matching receptor. Conclusion(s): Although both S-specific CD8 and CD4 T cell memory are established by prior infection, S-specific CD8 T cells predominated in blood after primary vaccination, with some clonotypes showing up to 1000-fold expansion across 1-2 mRNA doses. Vaccine-reactive CD8 clonotypes were present at the barrier nasal site after booster mRNA dosing. Severe disease imprinted a highly diverse S-reactive CD4 repertoire persisting through vaccination.

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity.

Recombinant adenovirus serotype 5 (Ad5) vector has been widely applied in vaccine development targeting infectious diseases, such as Ebola virus disease and coronavirus disease 2019 (COVID-19). However, the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines. Thus, there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors. Herein, we address this need by utilizing biocompatible nanoparticles to modulate Ad5-host interactions. We show that positively charged human serum albumin nanoparticles ((+)HSAnp), which are capable of forming a complex with Ad5, significantly increase the transgene expression of Ad5 in both coxsackievirus-adenovirus receptor-positive and -negative cells. Furthermore, in charge- and dose-dependent manners, Ad5/(+)HSAnp complexes achieve robust (up to 227-fold higher) and long-term (up to 60 days) transgene expression in the lungs of mice following intranasal instillation. Importantly, in the presence of preexisting anti-Ad5 immunity, complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity. These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

Mitochondrial enzymes infused into the kidney under hydrodynamic pressures significantly improves creatinine and BUN levels at the onset of moderate and severe acute kidney injuries

Introduction: The COVID-19 pandemic has highlighted the need to address how renal insults are treated. There is an urgent need to better understand the complex relationship between infections and kidney disease and develop safe and effective approaches that can be translated to the clinic. Hydrodynamic fluid delivery has shown promise in influencing renal function in disease models. This technique previously provided preconditioned protection in acute injury models by upregulating the mitochondrial adaptation, while hydrodynamic injections of saline alone have also improved microvascular perfusion. Accordingly, hydrodynamic mitochondrial gene delivery was applied to investigate its ability to halt renal impairment that may occur following episodes of acute moderate and severe injuries in a rat model. Methods: Transgene infusates were prepared by suspending approximately 2 μg of IDH2 (isocitrate dehydrogenase 2 (NADP+) and mitochondrial) plasmid DNA/g of body weight in 0.5 ml of saline. Animals were subjected to moderate (bilateral pedicle clamp 30 mins) or severe (bilateral pedicle clamp 60 mins) forms of ischemia-reperfusion injury (IRI). Infusates were delivered directly into the left renal vein within 5 seconds, roughly 1 hour after IRI was established. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were monitored for 2 weeks. Results: Significant reductions in the levels of both metabolites (p < 0.05 for both cases) were achieved with single transgene treatments administered at both time points. Specifically, the maximal rises in SCr and BUN levels were reduced by at least 50%, which translated the effects of a severe injury to a moderate injury and a moderate injury to a mild injury. Conclusions: Therefore, this study identifies an approach that boosts recovery and halts the progression of ischemia-reperfusion at its inception and can be vital for high-risk conditions and may help devise translation models to address the rising incidence of acute renal diseases. No conflict of interest

CILTACABTAGENE AUTOLEUCEL IN LENALIDOMIDE-REFRACTORY PATIENTS WITH PROGRESSIVE MULTIPLE MYELOMA AFTER 1-3 PRIOR LINES OF THERAPY: CARTITUDE-2 BIOLOGICAL CORRELATIVE ANALYSES AND UPDATED CLINICAL DATA

Background: Cohort A of the multicohort phase 2 CARTITUDE-2 (NCT04133636) study is assessing ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T-cell (CAR-T) therapy, in patients with multiple myeloma (MM) who received 1-3 prior lines of therapy (LOT) and were refractory to lenalidomide (len). This population is difficult to treat and has poor prognosis. Aims: To present updated results from CARTITUDE-2 Cohort A. Methods: All patients provided informed consent. Eligible patients had progressive MM after 1-3 prior LOT that included a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD). Patients were len-refractory and had no prior exposure to BCMA-targeting agents. Patients received a single cilta-cel infusion (target dose: 0.75×106 CAR+ viable T cells/kg) after lymphodepletion. Cilta-cel safety and efficacy were assessed. The primary endpoint was minimal residual disease (MRD) negativity at 10-5 by next generation sequencing. Patient management strategies were used to reduce the risk of movement and neurocognitive adverse events (MNTs). Other assessments included pharmacokinetic (PK) analyses (Cmax and Tmax of CAR+ T-cell transgene levels in blood), levels of cytokine release syndrome (CRS)-related cytokines (e.g., IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with immune effector cell-associated neurotoxicity syndrome (ICANS), and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: As of January 2022 (median follow-up: 17.1 months [range: 3.3-23.1]), cilta-cel was administered to 20 patients (male: 65%;median age: 60 years [range: 38-75]). Median number of prior LOT was 2 (range: 1-3);median time since MM diagnosis was 3.5 years (range: 0.7-8.0). 95% of patients were refractory to their last LOT;40% were triple-class refractory. Overall response rate was 95%, with 90% of patients achieving ≥complete response and 95% achieving ≥very good partial response. Median time to first response was 1.0 month (range: 0.7-3.3);median time to best response was 2.6 months (range: 0.9-13.6). All MRD-evaluable patients (n=16) achieved MRD negativity at 10-5. Median duration of response was not reached. The 12-month progression-free survival rate was 75% and the 12-month event-free rate was 79%. CRS occurred in 95% of patients (grade 3/4: 10%), with a median time to onset of 7 days (range: 5-9) and median duration of 3 days (range: 2-12). 30% of patients had neurotoxicity (5 grade 1/2 and 1 grade 3/4). ICANS occurred in 3 patients (15%;all grade 1/2);1 patient had facial paralysis (grade 2). No MNTs were observed. 1 death due to COVID-19 occurred and was assessed as treatment-related by the investigator;2 deaths due to progressive disease and 1 due to sepsis (not related to treatment) also occurred. Based on preliminary PK analyses of CAR transgene by qPCR, peak expansion of CAR-T cells occurred at day 10.5 (range: 8.7-42.9);median persistence was 153.5 days (range: 57.1-336.8). Summary/Conclusion: A single cilta-cel infusion led to deepening and durable responses at this longer follow-up (median 17.1 months) in patients with MM who had 1-3 prior LOT and were len-refractory. Follow-up is ongoing. We will present updated and detailed PK, cytokine, and CAR-T subset analyses as well as clinical correlation to provide novel insights into biological correlates of efficacy and safety in this difficult-to-treat patient population, which is being further evaluated in the CARTITUDE-4 study (NCT04181827;enrollment concluded).

Biological correlative analyses and updated clinical data of ciltacabtagene autoleucel (cilta-cel), a BCMA-directed CAR-T cell therapy, in lenalidomide (len)-refractory patients (pts) with progressive multiple myeloma (MM) after 1-3 prior lines of therapy (LOT): CARTITUDE-2, cohort A

Background: Cohort A of the multicohort phase 2 CARTITUDE-2 (NCT04133636) study is evaluating cilta-cel safety and efficacy in pts with MM who received 1-3 prior LOT and were len-refractory - a difficult- to-treat population with poor prognosis. We present updated results. Methods: Pts had progressive MM after 1-3 prior LOT, including a PI and IMiD, were len-refractory, and had no prior exposure to BCMA-targeting agents. A single cilta-cel infusion (target dose 0.75×106 CAR+ viable T cells/kg) was given post lymphodepletion. Safety and efficacy were assessed, and the primary endpoint was MRD negativity at 10-5. Management strategies were implemented to minimize risk of movement/neurocognitive AEs (MNTs). Pharmacokinetic (PK) analyses (Cmax and Tmax of CAR+ T-cell transgene levels in blood) are being conducted, as well as analyses of levels of CRS-related cytokines (eg, IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with ICANS, and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: As of January 2022 (median follow- up [MFU] 17.1 mo [range 3.3-23.1]), 20 pts (65% male;median age 60 y [range 38-75]) received cilta-cel. Pts received a median of 2 (range 1-3) prior LOT, and a median of 3.5 y (range 0.7-8.0) since MM diagnosis. 95% were refractory to last LOT, and 40% were triple-class refractory. ORR was 95%, 90% achieved CR or better, and 95% had ≥VGPR. Median times to first and best response were 1.0 mo (range 0.7-3.3) and 2.6 mo (range 0.9-13.6), respectively. 16 pts were MRDevaluable, all of whom achieved MRD negativity at 10-5. Median DOR was not reached and 12-mo event-free rate was 79%. The 12-mo PFS rate was 75%. Median time to onset of CRS was 7 d (range 5-9) and occurred in 95% of pts (gr 3/4: 10%), with median duration of 3 d (range 2-12). Neurotoxicity occurred in 30% of pts (5 gr 1/2;1 gr 3/4). 3 pts (15%) had ICANS (all gr 1/2);1 pt had gr 2 facial paralysis. No MNTs were seen. 1 death occurred due to COVID-19 (assessed as tx-related by the investigator), 2 due to progressive disease, and 1 due to sepsis (not related to tx). Preliminary PK analyses indicate that peak expansion of CAR-T cells occurred at d 10.5 (range 8.7-42.9) and median persistence was 153.5 d (range 57.1-336.8). Conclusions: At a longer MFU of 17.1 mo, a single cilta-cel infusion led to deepening and durable responses in pts with MM who had 1-3 prior LOT and were lenrefractory. Follow-up is ongoing. Updated and in-depth PK, cytokine, and CAR-T subset analyses and clinical correlation will be presented and provide novel insights into biological correlates of efficacy and safety in this pt population. This pt population is being further evaluated in the CARTITUDE-4 study (NCT04181827), which has concluded enrollment.

ASTHMA, AIRWAY INFLAMMATION AND PTSD ASSOCIATION: RELEVANT MODELS AND A ROLE OF IL17A

Background: There is a growing interest in airway inflammation and mental health. Recent genetic and epidemiological evidence supports an association between PTSD and asthma however, contributory immune mediators/mechanisms are unclear. Recent work from our group employs mouse aeroallergen, house dust mite (HDM) models to examine the role of severe asthma linked inflammatory T helper cells, Th17 and interleukin 17 (IL-17A) in regulating PTSD-relevant behaviors. Methods: A combination of behavioral, immunological, transgenic and transcriptomic approaches were used. 1) BALBc-C5a receptor treatment that shifts Th2 mild asthma phenotype to Th17/IL17a expansion and robust airway inflammation;2) IL-17a receptor knockout mice and 3) RNAseq transcriptomics of cortical and blood brain barrier compromised area, subfornical organ (SFO) tissue was performed. Fear conditioning and extinction was assessed as a PTSD-relevant behavior. Results: Induction of Th17/IL-17 in the BALBc/anti-C5aR1 treated mice resulted in compromised fear extinction and increased fear reinstatement. Absence of IL-17 signaling in IL17Ra deficient mice attenuated HDM effects on fear extinction. Preliminary evidence suggests a potential of the SFO in translating HDM effects to the medial prefrontal cortex, an area regulating fear extinction. Transcriptomic analyses revealed modulation of immune T cell-targeted signaling pathways within the SFO in mice with Th17A expansion. Conclusion: Overall, our work provides novel insights on mechanisms by which mediators of severe airway inflammation, Th17/IL17A regulate fear memory of relevance to PTSD. Beyond asthma-PTSD, our findings have relevant implications for other pulmonary (e.g. COVID-19) and autoimmune inflammatory conditions and mental health.

CRISPR-Cas: A continuously evolving technology

The discovery of the CRISPR/Cas microbial adaptive immune system and its ongoing development as a genome editing tool represents the work of many scientists around the world.The time line of CRISPR/Cas system shows that this technology is improving continuously to remove the demerits of preceding one with the aim of development of highly efficient, specific with low off target effect and ultimately transgene free technology in light of ethical and environmental issues related with transgenic technology.Initially, CRISPR/Cas9 was developed as method of choice as it provides targeted mutagenesis under in vivo condition and all the homeoalleles of a gene can be targeted in same plant, especially in case of polyploid species efficiently which is difficult through other existing technology.No residual or foreign gene insertion is required and modification is permanent.Now, CRISPR/Cpfl has been developed as more potent, efficient and simpler than CRISPR/Cas9.Different forms of Cas enzymes provide new avenues for regulation of genomic component.In view of the present devastating COVID-19 disaster the scientists used this novel technology for detection of virus in humans at an early stage of infection thus saving human lives.The evolution of CRlSPR'Cas technology, their advantages, apprehensions and solution, experimental design and updates of this technology is discussed in the present review. © 2021 Indian Council of Agricultural Research. All rights reserved.

AAVCOVID: A SINGLE-DOSE, THERMOSTABLE COVID-19 VACCINE with DURABLE IMMUNOGENICITY

Background: The SARS-CoV-2 pandemic has affected more than 250 million people worldwide resulting in 5 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses that easily delivered. Previously, we showed that an adeno-associated virus (AAV)-based vaccine candidate (AC1) elicited high humoral and cellular immunogenicity in mice and nonhuman primates (NHP) following a single injection, which provided near-sterilizing immunity against SARS-CoV-2 in NHP. Here, we developed optimized AAVCOVID vaccine candidates for higher potency and protection against variants of concern (VOC). Methods: The promoter in AC1 vector was substituted by three different promoters to increase the expression of Spike and they were tested in mice by single IM injection. Transgene expression and anti-Spike antibody and cellular responses were determined to assess vector potency. Then, the candidate that showed higher potency (ACM1) was engineered to express the Beta (ACM-Beta) and Delta (ACM-Delta) VOC Spike. The immunogenicity provided by ACM-Beta and ACM-Delta was characterized in mice and NHP. The cross-reactivity with the Wuhan and VOC Spikes was also assessed in the animals immunized with different Spike variants. Finally, challenge and durability studies were performed in NHPs vaccinated with the new candidates. Results: Vaccination with ACM1 candidate (miniCMV promoter) resulted in 100-fold higher Spike expression and 40-fold higher antibody responses compared to the prototypic AC1 candidate in mice. When ACM1, ACM-Beta and ACM-Delta were compared in mice, we found that the immune responses against the self-transgene were not significantly different. However, cross-reactivity was different, being ACM-Delta the candidate that better cross-neutralized the different VOC. Similar results were observed in NHP: higher potency of the candidates carrying the miniCMV promoter and similar cross-reactivity profiles. Additionally, ACM-Beta showed protection against Beta SARS-CoV-2 challenge and a durability study for ACM-Delta is ongoing. Conclusion: This work shows the adaptability and versatility of AAVCOVID vaccine platform to improve potency and protect against VOC. These observations together with the single, low dose requirement, high yield manufacturability, and 1-month stability for storage at room-temperature may make this technology well-suited to support effective immunization campaigns for emerging pathogens on a global scale.

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.

Identification of human CD4+ T cells specific for dominant epitopes of SARS-CoV-2 Spike and Nucleocapsid proteins with therapeutic potential

Introduction: Since December 2019, COVID-19 has spread rapidly across the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as patients after allogeneic haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both shortlived neutralising antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Methods: Activation of CD4+ T cells from 18 COVID-19 patients was determined by flow cytometry, both ex vivo and after in vitro restimulation with SARS-CoV-2 Spike and Nucleocapsid antigens. Immunodominant, 15-mer peptides were identified using epitope mapping. T cells clones specific for these epitopes were further chararacterised for the sensitivity and polarisation of their cytokine responses after in vitro restimulation, by ELISA and cytometric assay. Next-generation sequencing revealed fulllength, paired T Cell Receptor (TCR) αβ sequences. Results: We identified three patients with strong CD4+ T cells to SARSCoV- 2 antigens. From these patients, 81 T cell clones specific for a selection of 9 immunodominant epitopes (7 Spike and 2 Nucleocapsid epitopes) were generated. Cytokine analysis showed that the sensitivity and polarisation of T cell responses varied depending on the specific epitope. Moreover, TCRαβ sequences revealed an epitope-dependent difference in the level of clonality. Conclusions: We provide detailed information on SARS-CoV-2-specific CD4+ T cells, including their antigen-specificity, the nature of their cytokine responses and the full sequence of their TCRαβ. These cells have the potential to direct an effective immune response in COVID-19 patients. Our results form a crucial first step towards T cell therapy. Efforts are underway to develop transgenic CD4+ T cells that express the SARS-CoV- 2-specific TCRs identified.

Optimizing Commercial Manufacturing of Tisagenlecleucel for Patients in the US: A 4-Year Experiential Journey

Background: Tisagenlecleucel (Kymriah) is an autologous CD19-directed CAR-T-cell therapy, approved in Aug-2017 for treating children and young adults with relapsed/refractory (r/r) acute lymphoblastic leukemia and in May-2018 for treating adults with r/r diffuse large B-cell lymphoma. Post-approval, a key goal has been to upscale and continuously improve manufacturing success and turnaround time in the commercial settings to meet the needs of a global patient population. Here we report accrued experience from our 4-year journey of optimizing the commercial tisagenlecleucel manufacturing process at the US site (Morris Plains, NJ), for faster and successful delivery to patients in the US. Methods: As reported previously, the tisagenlecleucel manufacturing process includes leukapheresis of the patient's peripheral blood mononuclear cells, enrichment and activation of T cells, transduction of the lentiviral vector containing the anti-CD19 CAR transgene, activation with anti-CD3/CD28 antibody-coated beads, expansion in cell culture, washing, and formulation of the viable cells into a cryoformulation medium. The final product is then cryopreserved, shipped back to the treatment center and infused to patients (Tyagarajan, 2020). Use of cryopreserved leukapheresis material as the starting point in commercial manufacturing is unique to tisagenlecleucel;this allows flexibility in terms of scheduling leukapheresis when a patient's health is optimal to provide T cells, and also helps offset logistical challenges (Tyagarajan, 2019). Results: As of Jun-2021, tisagenlecleucel has been manufactured for >5000 patients worldwide, enabled by Novartis's significantly increased global manufacturing footprint at six sites strategically located across six countries (US, France, Switzerland, Germany, Japan and Australia) and a global treatment network of >340 certified centers, including 127 centers in the US. Specifically for the US manufacturing site, between Dec-2020 and Jun-2021, 376 patients in the US had starting material available for manufacturing. Overall, the manufactured product was available for shipment for 98% of patients (shipping success rate [SSR]). The commercial manufacturing success rate (MSR) was 96%, with an out-of-specification (OOS) rate of <3% and no OOS for viability. All ten OOS batches were released for infusion as benefit:risk assessment was positive. Manufacturing was cancelled for two patients upon physician's request. Immediate manufacturing capability without waiting time was available on receipt of all apheresis starting materials. The median time from start of manufacturing to shipping was 20 days. As is evident, the COVID-19 pandemic did not appear to have significantly affected the success rate or manufacturing turnaround time. These latest success metrics, reflecting significant improvements from 2018 to 2021 in MSR (69% to 96%), SSR (93% to 98%), and overall OOS rate (26% to 2%) including viability OOS rate (from 25% to 0%), are a result of upscaling the manufacturing capabilities, enhancements with hospitals focusing on optimizing apheresis collection and cryopreservation procedures, and continuous evaluation and improvement of the manufacturing process since tisagenlecleucel was first launched (Figure). Two key process and analytical improvements that were considered to have improved robustness of manufacturing and testing processes, reduced OOS rates, and minimized variability in turnaround time were introduced towards the end of 2020. Firstly, a simplified sample preparation procedure for final product cell count and viability measurement, which is more reflective of final product at infusion. Secondly, an alternate serum source (5% plasma-derived human AB serum [PD hABs]) which further improves process robustness with a trend towards improved growth and higher peak cell counts. Conclusions: Tisagenlecleucel's current global commercial manufacturing footprint and treatment network are well-positioned to meet anticipated future increase in demand for CAR-T therapies. Recent process improvements h ve significantly increased the MSR (to 96%) and SSR (to 98%), and immediate product availability for patients in need of CAR-T cells. Ongoing and upcoming process improvements are anticipated to further reduce the throughput time, thus allowing more patients faster access to CAR-T therapy. [Formula presented] Disclosures: Rodrigues: Novartis: Current Employment. Duran: Novartis: Current Employment. Eschgfaeller: Novartis: Current Employment. Kuzan: Novartis: Current Employment. Habucky: Novartis: Current Employment.