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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.

Transmission-blocking vaccines have the potential to be key contributors to malaria elimination. Such vaccines elicit antibodies that inhibit parasites during their development in Anopheles mosquitoes, thus breaking the cycle of transmission. To date, characterization of humoral responses to Plasmodium falciparum transmission-blocking vaccine candidate Pfs25 has largely been conducted in pre-clinical models. Here, we present molecular analyses of human antibody responses generated in a clinical trial evaluating Pfs25 vaccination. From a collection of monoclonal antibodies with transmission-blocking activity, we identify the most potent transmission-blocking antibody yet described against Pfs25; 2544. The interactions of 2544 and three other antibodies with Pfs25 are analyzed by crystallography to understand structural requirements for elicitation of human transmission-blocking responses. Our analyses provide insights into Pfs25 immunogenicity and epitope potency, and detail an affinity maturation pathway for a potent transmission-blocking antibody in humans. Our findings can be employed to guide the design of improved malaria transmission-blocking vaccines., Pfs25 is a transmission-blocking vaccine candidate for Plasmodium. Here, McLeod et al. analyze the antibody response to Pfs25 in sera from a clinical trial evaluating a Pfs25 vaccine candidate, identify a potent transmission-blocking antibody and determine recognized epitopes on Pfs25.

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016–2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.

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.

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.

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.