Summary V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo . Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction “cut-and-run” and suggest that it could be a significant cause of lymphocyte genome instability.

Transcription activator-like effector (TALE) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. Here, we systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only two-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

A newly identified process by which mistargeted V(D)J recombination could cause genome instability in childhood leukemia has been discovered. In this mechanism, called cut-and-run, the excised DNA byproducts of V(D)J recombination are rebound by the recombinase proteins and erroneously trigger double-strand breaks at multiple locations throughout the genome. Many of these breakpoints colocalize with known chromosome alterations in acute lymphoblastic leukemia (ALL).

Background Lefamulin (LEF) is a pleuromutilin antibiotic approved by the United States (US) FDA for the treatment of community-acquired bacterial pneumonia in adults. In addition to the potent antibacterial activity, LEF has demonstrated anti-inflammatory activity in an LPS induced lung neutrophilia model in mice. We investigated the anti-inflammatory activity of lefamulin in the H1N1 influenza mouse model in comparison to oseltamivir (OTV) and azithromycin (AZM). Methods Infection was performed in BALB/c mice by intranasal challenge ∼70 pfu influenza virus H1N1 A/PR/8/1934 (Day 0). Treatment with drugs at clinically relevant doses started on Day -1 (LEF 70 mg/kg/day and 110/140 mg/kg/day, s.c., AZM 30 mg/kg/day, i,p. and OTV 20 mg/kg/day p.o.) to Day 6. On Days 3 and 6, bronchioalveolar lavage fluid (BALF) was collected to measure infiltrating lung leukocytes and cytokines. Lung immunopathology following infection was evaluated on Day 6 by gross pathology at termination together with hematoxylin and eosin histopathology. Results In untreated vehicle control animals, the influenza infection progressed as expected with bodyweight loss, increased cell infiltration into the lung and increased levels of TNF-α, IL-6 at day 3 and 6. Treatment with LEF significantly decreased the total immune cell infiltration into the lung by day 6 at both doses tested (Figure Left). Cytokine levels in the BALF were significantly reduced on day 3 when the viral load peaked. Furthermore, LEF showed positive effects on lung gross pathology and survival. Oseltamivir and LEF, at both doses, appeared efficacious in the suppression of the development of influenza-induced bronchi-interstitial pneumonia, whereas azithromycin didn’t show reduced pathology (Figure Right). Anti-inflammatory effects of LEF, AZM and OTV in BALF of H1N1 infected mice infected with influenza virus H1N1. Left: Total lung leukocyte infiltrate following infection on day 0. Bars represent mean ± SEM. Right: Day 6 lung inflammation and degeneration histopathology score. Data are presented as mean histopathology score + SEM (n=10). One-way ANOVA, with Dunnett’s multiple comparisons against the vehicle control treatment was run for each timepoint. * represents significance level of p<0.05. ** indicates p<0.01. Conclusion Lefamulin showed anti-inflammatory treatment following acute influenza virus infection. Following influenza infection LEF was able to significantly reduce lung immunopathology and improve clinical outcome in mice. Results from this experiment were consistent with that observed in the LPS induced lung neutrophilia mouse model. Further studies are warranted to evaluate the immunomodulatory potential of Lefamulin. Disclosures Wolfgang W. Wicha, MSc, Nabriva Therapeutics: Grant/Research Support|Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds Sandy Kimber, PhD, Nabriva Therapeutics: Grant/Research Support Charlotte Cumper, BSc, Nabriva Therapeutics: Grant/Research Support Hon S. Lam, MSc, Nabriva Therapeutics: Grant/Research Support Lorena S. Ballesteros, MSc, Nabriva Therapeutics: Grant/Research Support Christopher Kirkham, PhD, Nabriva Therapeutics: Grant/Research Support Claire Richards, PhD, Nabriva Therapeutics: Grant/Research Support Steven P. Gelone, PharmD, Nabriva Therapeutics: Board Member|Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds|Nabriva Therapeutics: Stocks/Bonds Susanne Paukner, PhD, Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds.

Chimeric Antigen Receptor (CAR)-T cell therapy has achieved great success in treating a variety of liquid tumors, in particular CD19+ lymphomas. CAR technology is advancing rapidly, with notable improvements in efficacy, safety and complexity as human ingenuity seeks solid tumor applicability. Here we describe how to efficiently generate, expand and validate in-vitro and in-vivo efficacy of a clinically tested CD19-specific CAR. HLA-A*02:01+ CD3+ T-cells were transduced to express a CD19-CAR to >80% efficiency (in 5 donors) and reproducibly expanded to >68 fold over a 19 day period. In-vitro studies were used to assess tumor killing ability, utilizing a HLA-A*02:01+ patient derived xenograft (PDX) CD19+ cell line, alongside human cancer cell lines RAJI (CD19+) and K562 (CD19-). Analysis revealed the potent ability of CD19-CAR-T cells to specifically target CD19+ cells in a dose dependent manner. Supernatant analysis showed highly elevated IFNγ concentrations, identifying enhanced activation of CAR-T cells in the presence of CD19+ target cells only. In-vivo survival studies were completed to identify translatability & efficacy in both systems. Mice were inoculated with CD19+ cells to produce multiple tumor burdened models (RAJI, JEKO-1 & PDX4009). This demonstrated high efficacy of CAR-T at targeting both tumor cell line RAJI or PDX in vivo, with almost complete tumor regression seen in both models. JEKO-1 tumors saw progression slowed in comparison to non-transduced or no treatment controls however due to the nature of this cell line (highly tumorigenic) the level of tumor regression was not matched to the other models. Cell bio-distribution was assessed with CAR-T cells tracking successfully to the tumors as well as the highly vascularized tissues, which was expected and consistent with previous data. These assays can be translated to any CAR construct aimed at targeting either solid or hematological tumors and demonstrates an integrated platform for translating CAR research that can be utilized end to end. Citation Format: Lauren Kelsey, Karin Latta, Joey Kolb, Bincy John, Ilona Aylott, Daniel Rocca, Alexandru Bacita, Christopher Kirkham, Patrick Walters, Charlotte Humphery, Lorena Sueiro Ballesteros, Jezrom Self-Fordham, Louise Brackenbury, Chassidy Hall, Harris David, Julia Schueler, Robert Nunan. Functional and translatable efficacy of CAR-T cells targeting B cell lymphomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4097.