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Summary Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance., Graphical Abstract, Highlights • Methods to perform Cas9-mediated gene editing in primary human B cells are reported • Homologous recombination was confirmed by sequencing, PCR, and restriction digest • Replacement of B cell receptor loci in primary human B cells was achieved • Reprogramming the antibody specificity of B cells could have clinical importance, Molecular Biology; Immunology; Biotechnology; Cell Biology; Biological Sciences Research Methodologies

INTRODUCTION Most cancer patients die from cancer that recurs after spreading to a different tissue, rather than from their original tumor. After successful treatment of the original tumor, cancer cells that have disseminated to other sites can undergo dormancy, remaining viable but not proliferating. In breast, prostate, and other cancers, cancer cells can remain dormant and clinically undetectable for years and even decades before recurring, or awakening, as metastatic cancer. Little is known about what might initiate cancer awakening, and this in turn reduces our opportunities to prevent metastasis. RATIONALE Epidemiological studies have suggested that inflammation is linked to a higher risk of breast cancer recurrence after a period of clinical dormancy. Smoking, which causes chronic lung inflammation, is also associated with a higher risk of recurrence. However, whether inflammation can cause awakening is not clear. Inflammatory cells, such as neutrophils, can provide many different signals that promote cancer progression. Neutrophils can kill harmful microorganisms by the release of neutrophil extracellular traps (NETs) into the extracellular space. NETs are scaffolds of DNA with associated cytotoxic proteins and proteases [e.g., neutrophil elastase (NE) and matrix metalloproteinase 9 (MMP9)]. NETs induced by bacteria or by cancer cells can promote metastasis, but the mechanism by which this occurs is not known. In this study, we tested whether NETs formed during lung inflammation could induce awakening. RESULTS We found that sustained experimental lung inflammation—induced by either tobacco smoke exposure or nasal instillation of lipopolysaccharide (LPS)—converted dormant cancer cells to aggressive lung metastases in mice. Both types of sustained inflammation also caused the formation of NETs. Inhibiting NET formation or digesting the NETs’ DNA scaffold prevented conversion of single disseminated cancer cells to growing metastases in mouse models of breast and prostate cancer. The NET DNA bound to the extracellular matrix (ECM) protein laminin, thus bringing two NET-associated proteases, NE and MMP9, to their substrate. This in turn facilitated a sequential cleavage of laminin, first by NE and then by MMP9. The NET-mediated proteolytic remodeling of laminin revealed an epitope that triggered proliferation of dormant cancer cells through integrin activation and FAK/ERK/MLCK/YAP signaling. We generated a blocking antibody against NET-remodeled laminin, and this antibody prevented or reduced tobacco smoke exposure– or LPS-induced inflammation from awakening dormant cancer cells in mice. CONCLUSION Our data implicate NETs and NET-mediated ECM remodeling as critical mediators of inflammation-induced awakening in mouse models of dormancy. We propose that NETs awaken cancer by concentrating neutrophil proteases at the ECM protein laminin, allowing for sequential proteolytic remodeling of laminin and leading to integrin-mediated signaling in the cancer cells. Our findings set the stage for epidemiological studies to test possible links among inflammation or smoking, NETs, and recurrence after dormancy in human patients. If such links can be established, we envision that approaches similar to the ones used in mouse models in our study could be used to target NETs and their downstream effectors to reduce the risk of cancer recurrence in human patients.

Cancer immunotherapy can confer durable benefit, but the percentage of patients who respond to this approach remains modest. The ability to concentrate immunostimulatory compounds at the site of disease can overcome local immune tolerance and reduce systemic toxicity. Surgical resection of tumors may improve the efficacy of immunotherapy by removing the concentrated immunosuppressive microenvironment; however, it also removes tumor-specific leukocytes as well as tumor antigens that may be important to establishing antitumor immunity. Moreover, surgery produces a transient immunosuppressive state associated with wound healing that has been correlated with increased metastasis. Using multiple models of spontaneous metastasis, we show that extended release of agonists of innate immunity-including agonists of Toll-like receptor 7/8 (TLR7/8) or stimulator of interferon genes (STING)-from a biodegradable hydrogel placed in the tumor resection site cured a much higher percentage of animals than systemic or local administration of the same therapy in solution. Depletion and neutralization experiments confirmed that the observed prevention of local tumor recurrence and eradication of existing metastases require both the innate and adaptive arms of the immune system. The localized therapy increased the numbers of activated natural killer (NK) cells, dendritic cells, and T cells and induced production of large amounts of type I interferons, thereby converting an immunosuppressive post-resection microenvironment into an immunostimulatory one. The results suggest that the perioperative setting may prove to be a useful context for immunotherapy, particularly when the release of the therapy is extended locally.

Approximately 50% of the world's population is infected with Helicobacter pylori , leading to chronic inflammation, which increases the risk for gastric adenocarcinoma. MyD88 is a key adaptor molecule in inflammatory pathways involved in interleukin 1 (IL-1)/IL-18/Toll-like receptor signaling and has been shown to have divergent effects in carcinogenesis. The role of MyD88 in Helicobacter -induced gastric malignancy is unknown. Using a mouse model of Helicobacter -induced gastric cancer, we assessed the role of MyD88 in cancer development by evaluating gastric histopathology, apoptosis, proliferation, and cytokine expression. Infection of MyD88-deficient ( Myd88 −/− ) mice with Helicobacter resulted in early and rapid advancement to gastric dysplasia as early as 25 weeks postinfection. The progression of Helicobacter -induced disease to precancerous and cancerous lesions in the absence of MyD88 signaling was accompanied by increased gastric epithelial apoptosis and proliferation. In addition, inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-6, and IL-1β were highly expressed in association with the development of gastric dysplasia. These data suggest that MyD88 signaling retards development and progression to cancer during Helicobacter infection. This is the first study to show evidence of MyD88 protection in an infection-driven inflammation-associated cancer model.

Genome sequencing has revealed a large number of shared and personal somatic mutations across human cancers. In principle, any genetic alteration affecting a protein-coding region has the potential to generate mutated peptides that are presented by surface HLA class I proteins that might be recognized by cytotoxic T cells. To test this possibility, we implemented a streamlined approach for the prediction and validation of such neoantigens derived from individual tumors and presented by patient-specific HLA alleles. We applied our computational pipeline to 91 chronic lymphocytic leukemias (CLLs) that underwent whole-exome sequencing (WES). We predicted ∼22 mutated HLA-binding peptides per leukemia (derived from ∼16 missense mutations) and experimentally confirmed HLA binding for ∼55% of such peptides. Two CLL patients that achieved long-term remission following allogeneic hematopoietic stem cell transplantation were monitored for CD8(+) T-cell responses against predicted or confirmed HLA-binding peptides. Long-lived cytotoxic T-cell responses were detected against peptides generated from personal tumor mutations in ALMS1, C6ORF89, and FNDC3B presented on tumor cells. Finally, we applied our computational pipeline to WES data (N = 2488 samples) across 13 different cancer types and estimated dozens to thousands of predicted neoantigens per individual tumor, suggesting that neoantigens are frequent in most tumors.

Gastric cancer is the second most common cause of death from cancer worldwide, with approximately 800,000 deaths each year. The majority of human stomach tumors are associated with chronic infection with the bacterial pathogen Helicobacter pylori. In the U.S. gastric cancer incidences and mortality rates are higher among minority groups (African Americans, Asian/Pacific Islanders, Alaskan Natives, Hispanic-Latino) than whites. Accordingly, colonization rates of H. pylori are also higher in these groups with 75% for Alaska Natives, 62% for Mexican Americans, and 53% for non-Hispanic blacks compared to 26% for non-Hispanic whites, making H. pylori infection a disease that disproportionately afflicts minorities. The cause of this disparity is unknown. There is now considerable amount of confirmatory evidence that the host response to H. pylori is crucial in determining susceptibility to gastric cancer. Our objective was to study the role of the host innate immune response in Helicobacter disease progression to understand the existence of gastric cancer disparity. Specifically, we investigated the role of a key signaling molecule, myeloid differentiation primary response gene 88 (MyD88) in Helicobacter-induced gastric cancer. MyD88 is an adaptor protein that plays a central role in the innate and adaptive immune response. It is involved in inflammatory pathways of interleukin (IL) -1/IL-18 and Toll-like receptor signaling and is considered to be responsible for the chronic inflammation attributed to Helicobacter infection. Induction of chronic inflammation during infection with H. pylori is widely accepted to be the trigger for gastric carcinogenesis. We used a well-characterized animal model of gastric carcinogenesis that involves infection of C57BL/6 mice with H. felis, which is a close relative of the human pathogen H. pylori. Wild type (WT) and mice deficient in MyD88 (Myd88-/-) in the C57BL/6 background were infected with H. felis for 25 or 47 weeks and mouse stomachs processed for histology, immunohistochemistry, gene expression analysis, and apoptosis. Myd88-/- mice had a higher apoptotic index and progressed rapidly to neoplasia than WT mice. Microarray data showed differential gene expression between Helicobacter infected WT and Myd88-/- mice. At 25 weeks, Myd88-/- mice showed evidence of dysplasia, which was absent in WT mice. By 47 weeks, Myd88-/- mice had developed severe dysplasia, which is considered gastric cancer in situ while WT mice only showed early evidence of dysplasia. MyD88 deficiency therefore resulted in rapid progression to gastric dysplasia. In conclusion, our data show that in the absence of MyD88, infection with Helicobacter resulted in accelerated development of gastric cancer. These data suggest that the host immune response is a critical determinant of Helicobacter disease progression. Knowledge on the status of the immune responses during Helicobacter infection is the first step in understanding mechanisms of Helicobacter-induced carcinogenesis, which may lead to a better understanding of the existence of gastric cancer disparity. Citation Format: Kelly S. Doran, Ellese M. Carmona, Anirban Banerjee, Arnika Sharma, Marygorret Obonyo. Exploring the host immune response to understand the existence of Helicobacter-induced gastric cancer disparity. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B28.

Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages, known as pili, have been recently described in streptococcal pathogens, including GBS. The pilus tip adhesin, PilA, contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the host receptor and the contribution of PilA in central nervous system (CNS) disease pathogenesis are unknown. Here we show that PilA binds collagen, which promotes GBS interaction with the α2β1 integrin resulting in activation of host chemokine expression and neutrophil recruitment during infection. Mice infected with the PilA-deficient mutant exhibit delayed mortality, a decrease in neutrophil infiltration and bacterial CNS dissemination. We find that PilA-mediated virulence is dependent on neutrophil influx as neutrophil depletion results in a decrease in BBB permeability and GBS–BBB penetration. Our results suggest that the bacterial pilus, specifically the PilA adhesin, has a dual role in immune activation and bacterial entry into the CNS., Group B Streptococcus causes meningitis in newborn infants but how the bacterium crosses the blood-brain barrier is unknown. Here, the bacterial pili adhesion molecule, PilA, is shown to bind to collagen and promote binding of the bacteria to integrins expressed on the blood-brain endothelium.