Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer.

BACKGROUND: Natural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers in the context of an intact immune system, representing a valid cancer immunotherapy model. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. This study aimed to assess NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product. METHODS: Starting populations of CD5-depleted cells and PBMCs from healthy beagle donors were co-cultured for 14 days, phenotype, cytotoxicity, and cytokine secretion were measured, and samples were sequenced using the 3'-Tag-RNA-Seq protocol. Co-cultured human PBMCs and NK-isolated cells were also sequenced for comparative analysis. In addition, two first-in-dog clinical trials were performed in dogs with melanoma and osteosarcoma using autologous and allogeneic NK cells, respectively, to establish safety and proof-of-concept of this manufacturing approach. RESULTS: Calculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment. CONCLUSIONS: Overall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs.

Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Introduction: Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Experimental design: Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Results: Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Conclusion: Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

Low-dose mRNA-1273 COVID-19 vaccine generates durable memory enhanced by cross-reactive T cells.

Vaccine-specific CD4+ T cell, CD8+ T cell, binding antibody, and neutralizing antibody responses to the 25-µg Moderna messenger RNA (mRNA)­1273 vaccine were examined over the course of 7 months after immunization, including in multiple age groups, with a particular interest in assessing whether preexisting cross-reactive T cell memory affects vaccine-generated immunity. Vaccine-generated spike-specific memory CD4+ T cells 6 months after the second dose of the vaccine were comparable in quantity and quality to COVID-19 cases, including the presence of T follicular helper cells and interferon-γ­expressing cells. Spike-specific CD8+ T cells were generated in 88% of subjects, with equivalent memory at 6 months post-boost compared with COVID-19 cases. Lastly, subjects with preexisting cross-reactive CD4+ T cell memory exhibited stronger CD4+ T cell and antibody responses to the vaccine, demonstrating the biological relevance of severe acute respiratory syndrome coronavirus 2­cross-reactive CD4+ T cells.

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans.

Many unknowns exist about human immune responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. SARS-CoV-2-reactive CD4+ T cells have been reported in unexposed individuals, suggesting preexisting cross-reactive T cell memory in 20 to 50% of people. However, the source of those T cells has been speculative. Using human blood samples derived before the SARS-CoV-2 virus was discovered in 2019, we mapped 142 T cell epitopes across the SARS-CoV-2 genome to facilitate precise interrogation of the SARS-CoV-2-specific CD4+ T cell repertoire. We demonstrate a range of preexisting memory CD4+ T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Thus, variegated T cell memory to coronaviruses that cause the common cold may underlie at least some of the extensive heterogeneity observed in coronavirus disease 2019 (COVID-19) disease.

TFS and Spt4/5 accelerate transcription through archaeal histone-based chromatin.

RNA polymerase must surmount translocation barriers for continued transcription. In Eukarya and most Archaea, DNA-bound histone proteins represent the most common and troublesome barrier to transcription elongation. Eukaryotes encode a plethora of chromatin-remodeling complexes, histone-modification enzymes and transcription elongation factors to aid transcription through nucleosomes, while archaea seemingly lack machinery to remodel/modify histone-based chromatin and thus must rely on elongation factors to accelerate transcription through chromatin-barriers. TFS (TFIIS in Eukarya) and the Spt4-Spt5 complex are universally encoded in archaeal genomes, and here we demonstrate that both elongation factors, via different mechanisms, can accelerate transcription through archaeal histone-based chromatin. Histone proteins in Thermococcus kodakarensis are sufficiently abundant to completely wrap all genomic DNA, resulting in a consistent protein barrier to transcription elongation. TFS-enhanced cleavage of RNAs in backtracked transcription complexes reactivates stalled RNAPs and dramatically accelerates transcription through histone-barriers, while Spt4-Spt5 changes to clamp-domain dynamics play a lesser-role in stabilizing transcription. Repeated attempts to delete TFS, Spt4 and Spt5 from the T. kodakarensis genome were not successful, and the essentiality of both conserved transcription elongation factors suggests that both conserved elongation factors play important roles in transcription regulation in vivo, including mechanisms to accelerate transcription through downstream protein barriers.