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Over 80% of malaria-attributable deaths are in children under five. However, the only malaria vaccine recommended by the World Health Organization (WHO) for paediatric use, Mosquirix™, has limited efficacy. Complementary strategies, like monoclonal antibodies (mAbs), will be required to eradicate malaria. To discover new anti-malaria mAbs, we evaluated >28,000 antibody sequences from circulating B cells obtained from 45 Mosquirix™ vaccinees and selected 369 for testing. Many antibodies bound the circumsporozoite protein (CSP), a main surface protein on malaria and the malaria antigen in Mosquirix™, and several were exceptionally protective in mouse models of malaria. Through this work, we identified surprising correlations that suggest certain CSP sequences in Mosquirix™ may induce immunodominant antibody responses that dilute protective immunity. Further, we selected the antibodies most protective in preclinical mouse models and engineered them for improved manufacturability and developability to meet WHO guidelines. An optimised clinical candidate, MAM01, suitable for paediatric populations living in low-to-middle-income countries, was selected for clinical development.

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Vaccine development to prevent Salmonella Typhi infections has accelerated over the past decade, resulting in licensure of new vaccines, which use the Vi polysaccharide (Vi PS) of the bacterium conjugated to an unrelated carrier protein as the active component. Antibodies elicited by these vaccines are important for mediating protection against typhoid fever. However, the characteristics of protective and functional Vi antibodies are unknown. In this study, we investigated the human antibody repertoire, avidity maturation, epitope specificity, and function after immunization with a single dose of Vi-tetanus toxoid conjugate vaccine (Vi-TT) and after a booster with plain Vi PS (Vi-PS). The Vi-TT prime induced an IgG1-dominant response, whereas the Vi-TT prime followed by the Vi-PS boost induced IgG1 and IgG2 antibody production. B cells from recipients who received both prime and boost showed evidence of convergence, with shared V gene usage and CDR3 characteristics. The detected Vi antibodies showed heterogeneous avidity ranging from 10 μM to 500 pM, with no evidence of affinity maturation after the boost. Vi-specific antibodies mediated Fc effector functions, which correlated with antibody dissociation kinetics but not with association kinetics. We identified antibodies induced by prime and boost vaccines that recognized subdominant epitopes, indicated by binding to the de–O-acetylated Vi backbone. These antibodies also mediated Fc-dependent functions, such as complement deposition and monocyte phagocytosis. Defining strategies on how to broaden epitope targeting for S. Typhi Vi and enriching for antibody Fc functions that protect against typhoid fever will advance the design of high-efficacy Vi vaccines for protection across diverse populations.

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor repertoires in 12,591 HIV-1 Envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Acid stimulation in sandstone reservoirs containing significant amount of clays can end up with undesired results due to unexpected reactions between stimulation fluids and formation clays. This paper demonstrates how heavily damaged clay-rich sandstone reservoir completed with cased hole gravel pack (CHGP) in offshore Myanmar can be successfully established for commercial production with organic clay acid stimulation treatment. The formation is laminated dirty sand with very high clay content (up to 30%) and large gross height (>100m MD). Production logging results showed only a small portion of perforated intervals contributing to production. Thus, an appropriate stimulation treatment is required to unlock well potential and prevent screen failures from concentrated flow through a small interval. Given high clay content as well as presence of acid sensitive clays, conventional treatments using HCl as preflush and hydrofluoric (HF) acids as main fluids would result in potential damages from secondary and tertiary reactions. Furthermore, undissolved clays in the critical matrix left over from the treatment would potentially migrate and plug the pore throat. The new acid system was designed to generate small amount of HF in-situ (∼0.1%) at any given time with total strength of 1% HF, which would greatly minimize second and tertiary reactions and also permits acids travel deeper into the formation. Furthermore, the reaction products would react with the clays and physically "welding" the undissolved clays to the surface of the pore spaces permanently and prevent them from migration. The treatment was designed in three stages: 1) screen and gravel pack cleanup using coiled tubing (CT) jetting; 2) injectivity test; 3) main treatment consisting of acetic acids as preflush, and new acid system as main fluids followed by overflush. A newly designed linear gel containing relative permeability modifier was used for diversions. Two underperforming CHGP wells were treated, and both wells yielded 100% increase in productivity with no fine production observed at the surface. The success of the campaign owes to the sophisticated engineering workflow which starts from diagnostic of the damage zone and root-cause of the formation damage, followed by detailed analysis of various skin components using radial numerical reservoir modeling for all the reservoir layers that led to a proper treatment strategy and fluid design based on the damage and formation mineralogy as well as comprehensive laboratory tests. This has helped to minimize the risk of the treatment and eventually unlocked the production from the heavily damaged sandstone reservoir.

Small cell lung cancer (SCLC) is a devastating neuroendocrine carcinoma. MYCL (L-Myc) is frequently amplified in human SCLC, but its roles in SCLC progression are poorly understood. We isolated preneoplastic neuroendocrine cells from a mouse model of SCLC and found that ectopic expression of L-Myc, c-Myc, or N-Myc conferred tumor-forming capacity. We focused on L-Myc, which promoted pre-rRNA synthesis and transcriptional programs associated with ribosomal biogenesis. Deletion of Mycl in two genetically engineered models of SCLC resulted in strong suppression of SCLC. The high degree of suppression suggested that L-Myc may constitute a therapeutic target for a broad subset of SCLC. We then used an RNA polymerase I inhibitor to target rRNA synthesis in an autochthonous Rb/p53-deleted mouse SCLC model and found significant tumor inhibition. These data reveal that activation of RNA polymerase I by L-Myc and other MYC family proteins provides an axis of vulnerability for this recalcitrant cancer.

Background: The role of B cells and antibodies in anticancer immune responses may correlate with improved prognosis in several types of cancer. Indeed, tumor-reactive antibodies are detected in the blood of cancer patients, tumor-infiltrating B cells have been shown to produce tumor-reactive antibodies, and tumor-reactive antibodies can cause tumor regression in several mouse models. Taken together, these observations support further identification, isolation and characterization of antitumor antibodies from patients demonstrating effective anticancer responses and defining the cognate targets and mechanisms whereby they contribute to tumor control. Methods: We identified cohorts of patients with nonprogressing metastatic cancer who had received checkpoint immunotherapy and isolated their circulating plasmablasts. Antibody heavy and light chain paired sequences were obtained from individual cells using Atreca's Immune Repertoire Capture (IRCTM) technology. The expressed antibodies were then analyzed for their ability to bind to tumor cells as well as tumor tissue and their ability to mediate antitumor activity was explored in syngeneic mouse tumor models. Results: Elevated plasmablast levels were observed in individuals with nonprogressing metastatic cancer, and analysis of plasmablast antibody sequences revealed clonal families of B cells that persisted over time with hallmarks of affinity maturation and class switching. We also identified antibody sequences with features common to more than one patient, consistent with convergent antibody selection. In particular, one antibody (AB-213) isolated from a NSCLC patient was found to demonstrate binding to unrelated human tumors as well as the mouse EMT6 tumor. When AB-213 was expressed with a mouse IgG2a constant region the chimeric antibody showed efficacy in vivo by reducing tumor volume and increasing survival in Balb/c mice harboring the syngeneic EMT6 model. Antitumor activity of the chimeric antibody was observed to be dose-dependent when administered as monotherapy or in combination with checkpoint inhibitors. We feel, based on these data, AB-213 could become a very important clinical therapeutic. Citation Format: Gilson Baia, Amy Manning-Bog, Alexander Scholz, Jeff DeFalco, Michael Harbell, Danhui Zhang, Felix Chu, Beatriz Millare, May Sumi, Patricia Zuno, Judevin Lugar Sapugay, Dongkyoon Kim, Yvonne Leung, Shuwei Jiang, Xiaobin Tang, Kevin Williamson, Xiaomu Chen, Sean Carroll, Christine Dowd, Ish Dhawan, Jonathan Benjamin, Gregg Espiritu Santo, Nicole Haaser, Ngan Nguyen, Eldar Giladi, David Minor, Yann Chong Tan, Jeremy B. Sokolove, Lawrence Steinman, Tito Serafini, Guy Cavet, Norman M. Greenberg, Jacob Glanville, Wayne Volkmuth, Daniel E. Emerling, William H. Robinson. Mining the cancer immuno-responsome: The identification of functional antitumor antibodies from patients receiving checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3966.

Patient immune response to tumor can drive profound and durable clinical benefit, but the contribution of antibodies to this benefit is poorly understood. To further our understanding of the role of the humoral response, we compared antibody sequence repertoires of durable responder and non-responder melanoma patients before and during checkpoint inhibitor treatment. We collected pre- and on-treatment peripheral blood from 26 melanoma patients treated with pembrolizumab (9), ipilimumab (8) or nivolumab+ipilimumab (9). Of the 26 patients, 14 were durable responders (RECIST stable disease, partial response, or complete response for at least 6 months) and 12 were non-responders. We generated natively paired heavy and light chain antibody sequences from individual IgG plasmablasts (CD19+CD20-CD38+CD3-CD14-IgA-IgM-IgD-). Sequences were obtained from a total of 26,725 plasmablasts. These sequences were used to reconstruct lineages (sets of plasmablasts likely derived from a common progenitor B cell) where each lineage shares heavy and light V genes, CDR3 length and 80% sequence similarity in CDR3s. Durable responders exhibited an increase in somatic hypermutation (SHM) after initiation of treatment. In contrast, no significant change in SHM was observed in non-responders. Higher SHM was also observed in on-treatment repertoires of responders compared with those of non-responders suggesting that T cell checkpoint inhibitors promote activation of humoral immunity in clinical responders. Furthermore, persistent antibody lineages (observed both before and during treatment) showed higher SHM than lineages observed at only a single time point. Comparison of lineages between patients identified antibodies with high sequence similarity suggesting these antibodies may have arisen from convergent selection, i.e., different patients raising antibodies against shared or similar epitopes. These putative convergent antibodies were enriched in IgG2. IgG2 usage was also higher overall in durable responders than non-responders, even in samples taken prior to treatment. The higher levels of IgG2 observed in responders and in the potentially convergent sets of antibodies suggest that IgG2 antibodies may play a role in effective anti-tumor responses. In conclusion, our analysis of plasmablast repertoires from melanoma patients suggests that treatment with checkpoint inhibitors promotes SHM in durable responders, and that responder plasmablasts are enriched in IgG2. Moreover, patients make potentially convergent antibodies that are also enriched for the IgG2 subclass. Our observations demonstrate that the humoral immune response is remodeled in patients with anti-tumor responses and support a role for the humoral arm of the immune response in driving clinical benefit in patients treated with checkpoint inhibitors. Citation Format: Ngan Nguyen, Alusha Mamchak, Mariano Severgnini, Kevin S. Williamson, Xinqi Wu, Elliott F. Drabek, Michael Manos, Xiaomu Chen, Xiaobin Tang, Zoe Amiri, Chantia Carroll, Yvonne Leung, Dongkyoon Kim, Wayne Volkmuth, Norman Greenberg, Daniel Emerling, William H. Robinson, Guy Cavet, F. Stephen Hodi. Increased somatic hypermutation in the immunoglobulin sequences of melanoma patients who have durable response to checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 615.

This paper presents how to identify a hotspot, i.e. the most critical interval with a localized high gas rate and to determine the reliable maximum gas flow rate by interpreting production logging and calculating the fluid velocity at completion screen. This reliable characterization is highly important to prevent any failure of cased-hole gravel pack screens from fine particles. Generally, operators manage the gas well within allowable pressure draw down range. The rule of thumb for restriction on maximum recommended drawdown ranged from 500 psi to 1000 psi. However wells completed through sand control equipment completion might encounter sand face completion failure in the occurrence of extremely high velocity flowing hotspot zone near the screen or liner. Therefore it is necessary to pay close attention to the detection of hotspot zone using production logging interpretation. Moreover, calculation of the maximum gas rate based on gas velocity profile through the screen would determine the allowable maximum flow rate with advanced method. Normally In field cases, gas velocity higher than 1ft/s at the screen is considered as potential fluid velocity which could cause possible completion failure due to screen erosion accompanied by fine particles. One of the gas producing wells in SHWE project operated by Posco-Daewoo at Myanmar offshore, shows moderate mechanical skin damage and heterogeneous flow profile at the screen. Therefore rather than applying general drawdown criteria, calculating gas velocity at screen is recommended. To investigate the velocity profile at well bore, Production Logging was applied to Well-1 at SHWE field. By interpreting the Production logging, a highly biased flow profile was found out and reservoir interval showing highly biased flow would be suspected as of a hotspot zone. Production logging interpretation results indicate that the flow rate of 500 psig drawdown is much higher than the flow rate correspondent of 1ft/sec velocity at hotspot zone due to highly biased flow profile. In this case, the general drawdown criteria will not guarantee the sustainable maintenance of screen however exposes potential erosion to screen. Therefore, the gas wells suspected to have biased flow profile required to detect any potential hotspot areas and should be operated with advanced criteria to prevent potential sand face completion failure.

Primary cells are protected against oncogenic events by undergoing premature cellular senescence—an irreversible cell cycle arrest activated by mitogenic signaling as well as by overexpression of tumor suppressors, including p16INK4A, p53 and PML. In the human, downregulation of Dril1/E2F-BP1, a transcriptional regulator of E2F, promotes PML-dependent premature senescence and bypass of ant-iproliferative signaling by p19Arf/p53/p21Cip1 and p16INK4a to prevent both RasV12-induced and spontaneous senescence. The mouse ortholog, Arid3A/Bright, while highly characterized in B lymphocytes for its function in immunoglobulin transcription and hematopoiesis, had yet to be assessed for a function in growth control. That, along with the considerable sequence/exon structure diversion from its human orthologs, prompted us to evaluate Arid3a in this context. We report that reduction of Arid3a levels in B lymphocytes results in G1/S cell cycle arrest whereas overexpression of Arid3a leads to accumulation of Cyclin E, hyperphosphorylation of pRb, increased transcriptional activity of E2F1 and transformation in vivo. Arid3a associates with pRb in chromatin to release HDAC1 from the E2F1 promoter in proliferating cells. Arid3a mutants that fail to associate with pRb neither rescue senescence nor induce proliferation. Our results identify a function for Arid3 in cell cycle progression beyond its previously established role in immunoglobulin gene transcription.

Arid3a/Bright/Dril1 is a B cell-specific transactivator that regulates immunoglobulin heavy chain (IgH) gene transcription by binding promoter and enhancer-associated matrix attachment regions (MARs) within the IgH gene locus. Promoter MAR-mediated Arid3a transactivation is antagonized by direct competition of MAR binding by Cux1/CDP—a ubiquitously expressed repressor originally termed NF-μNR. We report that the NF-μNR complex includes Arid3a in B cells but not in non-B cells through mobility shift assays. The binding activity of NF-μNR and Arid3a in B cells is reciprocally altered during the cell division cycle and by the B cell mitogen lipopolysaccharide LPS. LPS treatment had no effect on Arid3a localization but increased its total abundance within the nucleus and cytoplasm. We show that this increased level of Arid3a is capable of displacing Cux from the MARs to facilitate IgH gene transcription. Finally, we showed that the MARs (termed Bf150 and Tx125) associated with the VH1 rearranged variable region expressed in the S107 murine plasmacytoma, can repress reporter gene transcription in non-B cells and that they can relieve the repression mediated by Eμ enhancer in B cells. These results have significant implications for early human development and demonstrate that MARs in IgH locus, NF-µNR and Arid3a regulate IgH gene expression in a concerted fashion. This paves the way for future studies examining the misregulation of this pathway in pediatric disease.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Multiple myeloma is a plasma cell neoplasm residing in bone marrow. Despite advances in myeloma therapies, novel therapies are required to improve patient outcomes. CD47 is highly expressed on myeloma cells and a potential therapeutic candidate for myeloma therapies. Flow cytometric analysis of patient bone marrow cells revealed that myeloma cells overexpress CD47 when compared with non-myeloma cells in 73% of patients (27/37). CD47 expression protects cells from phagocytosis by transmitting an inhibitory signal to macrophages. Here we show that blocking CD47 with an anti-CD47 monoclonal antibody increased phagocytosis of myeloma cells in vitro. In xenotransplantation models, anti-CD47 antibodies inhibited the growth of RPMI 8226 myeloma cells and led to tumor regression (42/57 mice), implicating the eradication of myeloma-initiating cells. Moreover, anti-CD47 antibodies retarded the growth of patient myeloma cells and alleviated bone resorption in human bone-bearing mice. Irradiation of mice before myeloma cell xenotransplantation abolished the therapeutic efficacy of anti-CD47 antibodies delivered 2 weeks after radiation, and coincided with a reduction of myelomonocytic cells in spleen, bone marrow and liver. These results are consistent with the hypothesis that anti-CD47 blocking antibodies inhibit myeloma growth, in part, by increasing phagocytosis of myeloma cells.

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.

e15161Background: Analyzing anti-cancer immune responses can offer insights into the mechanisms that underlie successful cancer therapies. We studied the B and T cell responses of 11 metastatic bre...

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing data sets are fragmented and difficult to analyze systematically. Here we present a pan-cancer resource and meta-analysis of expression signatures from ∼18,000 human tumors with overall survival outcomes across 39 malignancies. By using this resource, we identified a forkhead box MI (FOXM1) regulatory network as a major predictor of adverse outcomes, and we found that expression of favorably prognostic genes, including KLRB1 (encoding CD161), largely reflect tumor-associated leukocytes. By applying CIBERSORT, a computational approach for inferring leukocyte representation in bulk tumor transcriptomes, we identified complex associations between 22 distinct leukocyte subsets and cancer survival. For example, tumor-associated neutrophil and plasma cell signatures emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. This resource and associated analytical tools (http://precog.stanford.edu) may help delineate prognostic genes and leukocyte subsets within and across cancers, shed light on the impact of tumor heterogeneity on cancer outcomes, and facilitate the discovery of biomarkers and therapeutic targets.

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing datasets are fragmented and difficult to analyze systematically. We present a pan-cancer resource and comprehensive meta-analysis of expression signatures from ~18,000 human tumors with overall survival outcomes across 39 malignancies. While a third of prognostic genes were cancer-specific, a FOXM1 regulatory network is a major predictor of adverse outcomes, while favorably prognostic genes largely reflect tumor-associated leukocytes. Using a novel computational approach, we enumerated leukocyte subsets in bulk tumor transcriptomes, revealing complex novel malignancy-specific associations between 22 distinct leukocytes and cancer survival. Tumor-associated neutrophil-like polymorphonuclear cell and plasmacytic cells emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. Our results introduce new analytical tools for delineating prognostic genes and leukocytes within and across cancers, and shed light on the impact of tumour heterogeneity on cancer outcomes, with applications for discovering novel biomarkers and therapeutic targets Citation Format: Andrew J. Gentles, Aaron M. Newman, Chih Long Liu, Scott V. Bratman, Weiguo Feng, Dongkyoon Kim, Viswam S. Nair, Xu Yue, Amanda Khuong, Chuong D. Hoang, Maximilian Diehn, Robert B. West, Sylvia K. Plevritis, Ash A. Alizadeh. The prognostic landscape of genes and infiltrating immune cells across human cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR09.

Bright/Dril1/ARID3a is a B cell-specific, matrix association (or attachment) region-binding transcriptional regulator of immunoglobulin heavy chain genes and of E2F1-dependent cell cycle progression. Bright contains a central DNA binding domain termed ARID (AT-rich interacting domain) and a C-terminal region termed REKLES (for a conserved amino acid motif). The ARID domain has been identified in seven highly conserved families of metazoan proteins (ARID1-5 and JARID1-2), whereas REKLES is found only in the ARID3 subfamily (composed of Bright/ARID3a, Bdp/ARID3b, and Bright-like/ARID3c). REKLES consists of two subdomains: a modestly conserved N-terminal REKLESalpha and a highly conserved (among ARID3 orthologous proteins) C-terminal REKLESbeta. Previously we showed that Bright undergoes nucleocytoplasmic shuttling and that REKLESalpha and -beta were required, respectively, for nuclear import and Crm1-dependent nuclear export. Here we show that Bright further requires REKLESbeta for self-association or paralogue association and for nuclear matrix targeting. REK-LES promotes and regulates the extent of Bright multimerization, which occurs in the absence or presence of target DNA and is necessary for specific DNA binding. REKLESbeta-mediated interaction of Bright with Bdp, which localizes strictly to the nucleus, traps Bright within the nucleus via neutralization of its nuclear export activity. These results identify REKLES as a multifunctional domain that has co-evolved with and regulates functional properties of the ARID3 DNA binding domain.

Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.

Multiple myeloma is a plasma cell neoplasm characterized by frequent relapse following initial therapy, implying the presence of a therapy-resistant and tumor-initiating cell population. While tumor-initiating cells have been reported for human multiple myeloma, the data provide conflicting information regarding the surface immunophenotype of these cells. Here, we analyzed bone marrow myeloma and B-lineage cells of patients for fractionation, examined engraftment of myeloma cells in various mouse models, and finally investigated myeloma-initiating population by examining the repopulation of human myeloma cells in recipient mice. The clonality of repopulating cells was also determined by RT-PCR of immunoglobulin gene variable regions. First, the examination of immunophenotype of patients’ bone marrow cells allowed us to fractionate myeloma/plasma cell-enriching CD138+/CD38high plasmacytic cells and CD19+CD38low/− B lymphocytic cells in 82% (31/38) of human samples. Then, we transplanted patient bone marrow cells into RAG2−/−γc−/−, NOD/scid or NOD/scid/γc−/− mice with/without human fetal bone pieces and examined the engraftment of myeloma cells. While human bone-bearing mice engrafted myeloma/plasma cells of 10 out of 19 samples, none of 18 samples gave rise to the repopulation of clonal myeloma/plasma/B cells in human bone-free immunodeficient mice. Further analysis of fractionated cells demonstrated that CD38high/CD138+ cells led to production of clonal human antibodies (2/10 samples) and a repopulation of clonally-related CD19+CD38low or CD138+CD38+ B lineage cells (5/10 samples) in grafted human bone of recipient mice. However, CD19+CD38low/−CD138− cells did not engraft clonally-related cells in human bone-bearing mice (0/9 samples). The analysis of CD38high/CD138+ myeloma cells demonstrated that more than 70% of patients still have heterogeneous plasmacytic cells in the expressions of CD45, CD19 or CD27. Our results suggest that human bone marrow microenvironment may be required for the survival and the engraftment of primary myeloma cells into recipient mice and that CD38high/CD138+/− plasmacytic cells but not CD19+CD38low/− B cells enrich clonal myeloma cells engrafting into recipient mice which may be responsible for tumor-initiation of multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4315.

Background Butyrophillin-3A (BTN3A) three isoforms (3A1/3A2/3A3) are widely expressed on a variety of tumors.1 BTN3A1 plays a key role in phosphoantigen activation of Vg9Vd2 T-cells, key mediators of innate and adaptive anti-tumor immunity.2 Vg9Vd2 T-cell infiltration into tumor tissues is associated with a positive prognosis across multiple cancers,3 which makes BTN3A an interesting target for enhancing anti-tumor immunity. ImCheck Therapeutics is developing ICT01, an anti-BTN3A mAb that specifically activates Vg9Vd2 T-cells. ICT01 is currently in an international, multi-center Phase 1/2a clinical trial (NCT04243499, EVICTION Study). The level of BTN3A expression required for a clinical response to ICT01 is not known. Therefore, we developed novel immunohistochemistry (IHC) methods to enable a precision-medicine based approach to target population selection for dose escalation and potentially guiding patient selection in the expansion cohorts of the ongoing EVICTION study. Methods A panBTN3A IHC staining that recognizes the three isoforms was developed on Fresh frozen (FF) tissues, while BTN3A2- and BTN3A3-specific IHC methods were developed on formalin-fixed paraffin embedded (FFPE) tissues. BTN3A1-specific staining is still under development. Transfected knock-out/knock-in cell lines and positive tissues were used to assess antibody specificity. BTN3A expression was then analyzed on both normal and associated tumor tissue using tissue microarrays (TMA) and selected frozen blocks from tumor biopsies. FACS analyses were also performed on dissociated lung and pancreatic cancer biopsies to determine BTN3A (3 isoforms) membrane expression on tumor-infiltrating immune cells and cancer/stromal cells. Results In normal tissues, BTN3A2 and BTN3A3 specific IHC signals were granular cytoplasmic in epithelial cells, with positive mononuclear and endothelial cells. Higher expression in lung, colon, and small intestine tissues was observed. Regarding panBTN3A expression, inter-indication and inter-patient heterogeneity was observed among head and neck, lung, melanoma, bladder, colon, pancreas, breast, and prostate cancer tissues, with both cytoplasmic and membranous localizations. The major finding was higher expression of BTN3A2 on malignant cells in melanoma, lung, colon, and prostate cancers, as compared to normal tissue. Finally, FACS analyses of lung and pancreatic cancer tissues revealed stronger expression of all BTN3A isoforms at the cell surface of infiltrated immune cells compared to its expression on stromal cells. Conclusions These validated IHC methods supported the selection of cancer indications for the EVICTION trial and will potentially help identify specific tumor subtypes and patients that will most likely benefit from ICT01 treatment. References Compte E, Pontarotti P, Collette Y, Lopez M, Olive D. Frontline: characterization of BT3 molecules belonging to the B7 family expressed on immune cells. Eur J Immunol 2004;34:2089–99. Juan-Luis Blazquez, Audrey Benyamine, Christine Pasero, and Daniel Olive. New Insights into the Regulation of ?d T Cells by BTN3A and Other BTN/BTNL in Tumor Immunity. Front Immunol 2018;9:1601. Andrew J. Gentles, Aaron M. Newman, Chih Long Liu, Scott V. Bratman, Weiguo Feng, Dongkyoon Kim, Viswam S. Nair, Yue Xu, Amanda Khuong, Chuong D. Hoang, Maximilian Diehn, Robert B. West, Sylvia K. Plevritis, Ash A. Alizadeh. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med 2015 Aug; 21(8):938–945.

Background We have previously described ATRC-101, a fully human, engineered IgG1 antibody binding a tumor-restricted ribonucleoprotein (RNP) complex as its target. ATRC-101 is currently under evaluation in the clinic as a monotherapy for solid tumors. Following target engagement, ATRC-101 functions in an Fc-mediated fashion to deliver the target to the innate immune system, which modifies the tumor microenvironment and generates an adaptive immune response involving CD8+ T cells leading to anti-tumor activity in syngeneic mouse models. Binding of ATRC-101 appears restricted to malignant tissues in both mouse models and human, across a range of cancer histologic phenotypes, including carcinomas that are known candidates for anti-PD-1 treatment. In the EMT6 mouse model, representing a T cell-excluded phenotype in which anti-PD-1 agents display limited activity, ATRC-101 monotherapy was uniformly vigorous with persistent anti-tumor memory. When co-administered at a lower dose with anti-PD-1, the combination of therapy demonstrated a robust and heightened anti-tumor response relative to either agent dosed as monotherapy at similar concentrations. Methods To gain insight into the mechanisms that contribute to the anti-tumor effect with combination therapy, in vivo experiments in the EMT6 syngeneic mouse model were performed to determine temporal and spatial patterns of infiltrates and assessed tumors by using whole exome sequencing following administration of ATRC-101 vs. vehicle control. Within naive human tumor samples, coincident immunoreactivities of ATRC-101 and PD-L1 were also characterized. Results In mice treated with ATRC-101, analysis by immunofluorescence revealed a significant increase in the percentage of PD-1 reactive T cells within the tumor microenvironment. Elevated transcripts for PD-L1 also were detected in tumors from mice administered ATRC-101 vs baseline levels or vehicle control. When human tumor tissues were characterized for coincident expression of these targets, a high prevalence of ATRC-101 immunoreactivity was noted in both PD-L1 reactive and non-reactive tumor cores. Across multiple indications, ATRC-101 immunoreactivity was apparent in > 50% of PD-L1+ cores. Conclusions In situ studies suggest the target of ATRC-101 may co-locate with PD-L1, and in vivo studies indicate that ATRC-101 administration increases PD-L1 transcripts and PD-1-positive infiltrates in mouse tumor. Altogether, our data support studies to combine ATRC-101 with agents targeting PD-1 in the clinical treatment of solid tissue malignancies. Acknowledgements We acknowledge the significant effort and contributions of our colleagues from the clinical, in vivo pharmacology, translational sciences, in vitro pharmacology, and cell biology groups. This includes Mark Armanini, Erin Brosey, Chantia Carroll, Sean M. Carroll, Nicole Haaser, Benjamin Haugen, Dongkyoon Kim, Beatriz Millare, Yann Chong Tan, Danhui Zhang, and Patricia Zuno. Trial Registration NCT04244552 Ethics Approval The study was approved by WIRB (Western Institutional Review Board) on Jun 11, 2013. The WIRB study number is 20130121. Reference DeFalco J, Harbell M, Manning-Bog A, et al. Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens. Clinical Immunology 2018; 187:37–45.