Prevalence of established and emerging biomarkers of immune checkpoint inhibitor response in advanced hepatocellular carcinoma.

The clinical deployment of immune checkpoint inhibitors (ICIs) has created a tandem drive for the identification of biomarkers linked to benefit. Comprehensive genomic profiling was performed to evaluate the frequency of genomic biomarkers of ICI response in 755 patients with advanced hepatocellular carcinoma (HCC). Median age was 62 years' old, 73% were male, 46% had extrahepatic disease, 107 had documented hepatitis C, 96 had hepatitis B and 4 patients were coinfected. Median tumor mutation burden (TMB) was 4 mutations/Mb and only 6 tumors (0.8%) were TMB-high. Out of 542 cases assessed for microsatellite instability (MSI), one (0.2%) was MSI-high and TMB-high. Twenty-seven (4%) patients had POLE/D alterations. One patient had a pathogenic POLE R762W mutation but TMB was 4 mutations/Mb. Forty percent had DNA damage response gene alterations. In a small case series (N=17) exploring the relationship between biomarkers and ICI response, one patient (TMB 15 mutations/Mb, MSI-low) had a sustained complete response to nivolumab lasting > 2 years. Otherwise there were no significant genomic or TMB differences between responders, progressors, and those with stable disease. Overall, markers of genomic instability were infrequent in this cohort. Larger clinically annotated datasets are needed to explore genomic and non-genomic determinants of ICI response in HCC

IgA as a potential candidate for enteric monoclonal antibody therapeutics with improved gastrointestinal stability

Mucosal surfaces of the gastrointestinal tract play an important role in immune homeostasis and defense and may be compromised by enteric disorders or infection. Therapeutic intervention using monoclonal antibody (mAb) offers the potential for treatment with minimal off-target effects as well as the possibility of limited systemic exposure when administered orally. Critically, to achieve efficacy at luminal surfaces, mAb must remain stable and functionally active in the gastrointestinal environment. To better understand the impact of isotype, class, and molecular structure on the intestinal stability of recombinant antibodies, we used an in vitro simulated intestinal fluid (SIF) assay to evaluate a panel of antibody candidates for enteric mAb-based therapeutics. Recombinant IgG1 was the least stable following SIF incubation, while the stability of IgA generally increased upon polymerization, with subtle differences between subclasses. Notably, patterns of variability within and between mAbs suggest that variable regions contribute to mAb stability and potentially mediate mAb susceptibility to proteases. Despite relatively rapid degradation in SIF, mAbs targeting Enterotoxigenic Escherichia coli (ETEC) displayed functional activity following SIF treatment, with SIgA1 showing improved function compared to SIgA2. The results of this study have implications for the design of enteric therapeutics and subsequent selection of lead candidates based upon in vitro intestinal stability assessments

Immune features that afford protection from clinical disease versus sterilizing immunity to Bordetella pertussis infection in a nonhuman primate model of whooping cough

The respiratory bacterial infection caused by Bordetella pertussis (whooping cough) is the only vaccine-preventable disease whose incidence has been increasing over the last 3 decades. To better understand the resurgence of this infection, a baboon animal model of pertussis infection has been developed. Naïve baboons that recover from experimental pertussis infection are resistant both to clinical disease and to airway colonization when re-challenged. In contrast, animals vaccinated with acellular pertussis vaccine and experimentally challenged do not develop disease, but airways remain colonized for 4-6 weeks. We explored the possibility that the IgG antibody response to pertussis infection is qualitatively different from antibodies induced by acellular pertussis vaccination. IgG was purified from pertussis-convalescent baboons shown to be resistant to pertussis disease and airway colonization. Purified IgG contained high titers to pertussis toxin, pertactin, and filamentous hemagglutinin. This pertussis-immune IgG or control IgG was passively transferred to naïve, juvenile baboons before experimental airway pertussis inoculation. The control animal that received normal IgG developed a typical symptomatic infection including leukocytosis, cough and airway colonization for 4 weeks. In contrast, baboons that received convalescent IgG maintained normal WBC counts and were asymptomatic. However, despite remaining asymptomatic, their airways were colonized for 4-6 weeks with B. pertussis. All animals developed IgG and IgA anti-pertussis antibody responses. Interestingly, the clearance of B. pertussis from airways coincided with the emergence of a serum anti-pertussis IgA response. These studies demonstrate that passive administration of pertussis-specific IgG from previously infected animals can prevent clinical disease but does not affect prolonged airway colonization with B. pertussis. This outcome is similar to that observed following acellular pertussis vaccination. Understanding immune mechanisms—other than IgG—that are capable of preventing airway colonization with B. pertussis will be critical for developing more effective vaccines to prevent whooping cough

Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis Against Enterotoxigenic Escherichia coli Infection

Background. Enterotoxigenic Escherichia coli (ETEC) cause diarrheal illness in infants in the developing world and travelers to endemic countries including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Methods. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Results. Over 300 unique anti-CfaE IgG1 HuMabs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or secretory IgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A two to four log decrease of colony forming units was observed as compared to irrelevant isotype controls. Conclusions. We identified fully human monoclonal antibodies against CfaE adhesion domain that can be potentially employed as an immunoprophylaxis to prevent ETEC-related diarrhea

Identification of a Human Monoclonal Antibody to Replace Equine Diphtheria Anti-toxin for the Treatment of Diphtheria

Diphtheria anti-toxin (DAT) has been used to treat Corynebacterium diphtheriae infection for over one hundred years. While the global incidence of diphtheria has declined in the 20th century, the disease remains endemic in many parts of the world and significant outbreaks still occur. Diphtheria anti-toxin is an equine polyclonal antibody with considerable side effects that is in critically short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we cloned human monoclonal antibodies (HuMabs) directly from antibody secreting cells of human volunteers immunized with Td vaccine. We isolated a diverse panel of HuMabs that recognized diphtheria toxoid and recombinant protein fragments of diphtheria toxin. Forty-one unique HuMabs were expressed in 293T cells and tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays. The lead candidate HuMab, 315C4 potently neutralized diphtheria toxin with an EC50 of 0.65 ng/mL. Additionally, 25 μg of 315C4 completely protected guinea pigs in an in vivo lethality model. In comparison, 1.6 IU of DAT was necessary for full protection resulting in an estimated relative potency of 64 IU/mg for 315C4. We further established that our lead candidate HuMab binds to the receptor binding domain of diphtheria toxin and blocks the toxin from binding to the putative receptor, heparin binding-epidermal growth factor like growth factor. The discovery of a specific and potent neutralizing antibody against diphtheria toxin holds promise as a potential human therapeutic and is being developed for human use

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naive yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer

Novel roles for class II Phosphoinositide 3-Kinase C2 beta in signalling pathways involved in prostate cancer cell invasion

Phosphoinositide 3-kinases (PI3Ks) regulate several cellular functions such as proliferation, growth, survival and migration. The eight PI3K isoforms are grouped into three classes and the three enzymes belonging to the class II subfamily (PI3K-C2a, ß and ?) are the least investigated amongst all PI3Ks. Interest on these isoforms has been recently fuelled by the identification of specific physiological roles for class II PI3Ks and by accumulating evidence indicating their involvement in human diseases. While it is now established that these isoforms can regulate distinct cellular functions compared to other PI3Ks, there is still a limited understanding of the signalling pathways that can be specifically regulated by class II PI3Ks. Here we show that PI3K-C2ß regulates mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) activation in prostate cancer (PCa) cells. We further demonstrate that MEK/ERK and PI3K-C2ß are required for PCa cell invasion but not proliferation. In addition we show that PI3K-C2ß but not MEK/ERK regulates PCa cell migration as well as expression of the transcription factor Slug. These data identify novel signalling pathways specifically regulated by PI3K-C2ß and they further identify this enzyme as a key regulator of PCa cell migration and invasion

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system

Warmer Weather Linked to Tick Attack and Emergence of Severe Rickettsioses

The impact of climate on the vector behaviour of the worldwide dog tick Rhipicephalus sanguineus is a cause of concern. This tick is a vector for life-threatening organisms including Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, R. conorii, the agent of Mediterranean spotted fever, and the ubiquitous emerging pathogen R. massiliae. A focus of spotted fever was investigated in France in May 2007. Blood and tissue samples from two patients were tested. An entomological survey was organised with the study of climatic conditions. An experimental model was designed to test the affinity of Rh. sanguineus for biting humans in variable temperature conditions. Serological and/or molecular tools confirmed that one patient was infected by R. conorii, whereas the other was infected by R. massiliae. Dense populations of Rh. sanguineus were found. They were infected with new genotypes of clonal populations of either R. conorii (24/133; 18%) or R. massiliae (13/133; 10%). April 2007 was the warmest since 1950, with summer-like temperatures. We show herein that the human affinity of Rh. sanguineus was increased in warmer temperatures. In addition to the originality of theses cases (ophthalmic involvements, the second reported case of R. massiliae infection), we provide evidence that this cluster of cases was related to a warming-mediated increase in the aggressiveness of Rh. sanguineus, leading to increased human attacks. From a global perspective, we predict that as a result of globalisation and warming, more pathogens transmitted by the brown dog tick may emerge in the future