Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti-PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies.

Immune checkpoints are emerging as novel targets for cancer therapy, and antibodies against them have shown remarkable clinical efficacy with potential for combination treatments to achieve high therapeutic index. This work aims at providing a novel approach for the generation of several novel human immunomodulatory antibodies capable of binding their targets in their native conformation and useful for therapeutic applications. We performed a massive parallel screening of phage libraries by using for the first time activated human lymphocytes to generate large collections of single-chain variable fragments (scFvs) against 10 different immune checkpoints: LAG-3, PD-L1, PD-1, TIM3, BTLA, TIGIT, OX40, 4-1BB, CD27 and ICOS. By next-generation sequencing and bioinformatics analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment. As a proof of concept of the quality/potency of the binders identified by this approach, human IgGs from three of these collections (i.e., PD-1, PD-L1 and LAG-3) were generated and shown to have comparable or better binding affinity and biological activity than the clinically validated anti-PD-1 mAb nivolumab. The repertoires generated in this work represent a convenient source of agonistic or antagonistic antibodies against the 'Checkpoint Immunome' for preclinical screening and clinical implementation of optimized treatments.

Chronic hepatitis C viral infection subverts vaccine-induced T-cell immunity in humans.

UNLABELLED: Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high-magnitude, durable CD4(+) and CD8(+) T-cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV-specific immune responses and determine T-cell cross-reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1-infected patients were vaccinated using heterologous adenoviral vectors (ChAd3-NSmut and Ad6-NSmut) encoding HCV NS proteins in a dose escalation, prime-boost regimen, with and without concomitant pegylated interferon-α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon-γ enzyme-linked immunospot assays. Cross-reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV-specific T-cell responses following vaccination was markedly reduced. CD8(+) HCV-specific T-cell responses were detected in 15/24 patients at the highest dose, whereas CD4(+) T-cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T-cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. CONCLUSION: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T-cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections.

Mucosal delivery of a vectored RSV vaccine is safe and elicits protective immunity in rodents and nonhuman primates.

Respiratory Syncytial Virus (RSV) is a leading cause of severe respiratory disease in infants and the elderly. No vaccine is presently available to address this major unmet medical need. We generated a new genetic vaccine based on chimpanzee Adenovirus (PanAd3-RSV) and Modified Vaccinia Ankara RSV (MVA-RSV) encoding the F, N, and M2-1 proteins of RSV, for the induction of neutralizing antibodies and broad cellular immunity. Because RSV infection is restricted to the respiratory tract, we compared intranasal (IN) and intramuscular (M) administration for safety, immunogenicity, and efficacy in different species. A single IN or IM vaccination completely protected BALB/c mice and cotton rats against RSV replication in the lungs. However, only IN administration could prevent infection in the upper respiratory tract. IM vaccination with MVA-RSV also protected cotton rats from lower respiratory tract infection in the absence of detectable neutralizing antibodies. Heterologous prime boost with PanAd3-RSV and MVA-RSV elicited high neutralizing antibody titers and broad T-cell responses in nonhuman primates. In addition, animals primed in the nose developed mucosal IgA against the F protein. In conclusion, we have shown that our vectored RSV vaccine induces potent cellular and humoral responses in a primate model, providing strong support for clinical testing.

A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory.

A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8(+) and CD4(+) HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.

Fusion of HCV nonstructural antigen to MHC class II-associated invariant chain enhances T-cell responses induced by vectored vaccines in nonhuman primates.

Despite viral vectors being potent inducers of antigen-specific T cells, strategies to further improve their immunogenicity are actively pursued. Of the numerous approaches investigated, fusion of the encoded antigen to major histocompatibility complex class II-associated invariant chain (Ii) has been reported to enhance CD8(+) T-cell responses. We have previously shown that adenovirus vaccine encoding nonstructural (NS) hepatitis C virus (HCV) proteins induces potent T-cell responses in humans. However, even higher T-cell responses might be required to achieve efficacy against different HCV genotypes or therapeutic effect in chronically infected HCV patients. In this study, we assessed fusion of the HCV NS antigen to murine and human Ii expressed by the chimpanzee adenovirus vector ChAd3 or recombinant modified vaccinia Ankara in mice and nonhuman primates (NHPs). A dramatic increase was observed in outbred mice in which vaccination with ChAd3 expressing the fusion antigen resulted in a 10-fold increase in interferon-γ(+) CD8(+) T cells. In NHPs, CD8(+) T-cell responses were enhanced and accelerated with vectors encoding the Ii-fused antigen. These data show for the first time that the enhancement induced by vector vaccines encoding li-fused antigen was not species specific and can be translated from mice to NHPs, opening the way for testing in humans.

Role of IL28B gene polymorphism and cell-mediated immunity in spontaneous resolution of acute hepatitis C.

BACKGROUND: A single-nucleotide polymorphism (SNP; rs12979860) near the IL28B gene has been associated with spontaneous and treatment-induced hepatitis C virus clearance. We investigated predictors of spontaneous disease resolution in a cohort of patients with acute hepatitis C (AHC), analyzing epidemiological, clinical and virological parameters together with IL28B.rs12979860 genotypes and cell-mediated immunity (CMI). METHODS: Fifty-six symptomatic AHC patients were enrolled and followed prospectively. CMI was measured in 31 patients at multiple time points by interferon-γ enzyme-linked immunospot assay and was correlated to the IL28B.rs12979860 SNP. RESULTS: Eighteen patients had a self-limiting AHC that was associated with female sex (P = .028), older age (P = .018), alanine aminotransferase level >1000 U/L (P = .027), total bilirubin level >7 mg/dL (P = .036), and IL28B.rs12979860 genotype CC (P = .030). In multivariate analysis, only CC genotype was independently associated with self-limiting AHC (odds ratio, 5.3; 95% confidence interval, 1.1-26.5). Patients with the CC genotype with self-limiting AHC had a stronger (P = .02) and broader (P = .013) CMI than patients with the CT genotype with chronically evolving AHC. In patients with chronically evolving disease, CC genotype was associated with a broader CMI compared to CT genotype (P = .028). A negative CMI was more frequently associated with CT genotype among persistently infected patients (P = .043) and with persistent infection among CT patients (P = .033). CONCLUSIONS: . Self-limiting AHC was independently associated with CC genotype. The correlation between IL28B.rs12979860 genotypes and CMI is suggestive of a possible important role of CMI in favoring hepatitis C virus clearance in CC patients.

Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man.

Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species.

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.