Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/ 68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.hemagglutinin | protein design | bacterial expression

Antigen-presenting cells (APCs) occupy diverse anatomical tissues, but their tissue-restricted homeostasis remains poorly understood. Here, working in mouse models of inflammation, we found that mTOR-dependent metabolic adaptation was required at discrete locations. mTOR was dispensable for DC homeostasis in secondary lymphoid tissues but necessary to regulate cellular metabolism and accumulation of CD103+ DCs and alveolar macrophages in lung. Moreover, while numbers of mTOR-deficient lung CD11b+ DCs were not changed, they were metabolically reprogrammed to skew allergic inflammation from eosinophilic Th2 to neutrophilic Th17 polarity. The mechanism for this change was independent of translational control but dependent on inflammatory DC, which produced IL-23 and increased fatty acid oxidation. mTOR therefore mediates metabolic adaptation of APCs in distinct tissues, influencing the immunological character of allergic inflammation.

The hemagglutinin protein (HA) on the surface of influenza virus is essential for viral entry into the host cells. The HA1 subunit of HA is also the primary target for neutralizing antibodies. The HA2 subunit is less exposed on the virion surface and more conserved than HA1. We have previously designed an HA2-based immunogen derived from the sequence of the H3N2 A/HK/68 virus. In the present study, we report the design of an HA2-based immunogen from the H1N1 subtype (PR/8/34). This immunogen (H1HA0HA6) and its circular permutant (H1HA6) were well folded and provided complete protection against homologous viral challenge. Antisera of immunized mice showed cross-reactivity with HA proteins of different strains and subtypes. Although no neutralization was observable in a conventional neutralization assay, sera of immunized guinea pigs competed with a broadly neutralizing antibody, CR6261, for binding to recombinant Viet/04 HA protein, suggesting that CR6261-like antibodies were elicited by the immunogens. Stem domain immunogens from a seasonal H1N1 strain (A/NC/20/99) and a recent pandemic strain (A/Cal/07/09) provided cross-protection against A/PR/8/34 viral challenge. HA2-containing stem domain immunogens therefore have the potential to provide subtype-specific protection. Influenza virus, the causative agent of flu, is responsible for yearly epidemics and frequent pandemics around the world. The virus changes its genetic makeup constantly to escape the immune pressure from the host, causing fresh epidemics. The envelope of the virus has two major glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA is a trimer of HA1 and HA2 dimers that are produced by cleavage of the precursor HA0. The globular head domain of the protein is composed exclusively of HA1 and is involved in binding of the virus to host cell sialic acid receptors leading to endosomal uptake of the virus into the cell. HA2, along with regions of HA1, forms the membrane-proximal stalk that is in a metastable conformation, poised to change its conformation upon exposure to the low pH of the endosomes. This conformational change brings about fusion of viral and host endosomal membranes and release of the viral contents into the cytoplasm (25).Antibodies (Abs) generated against the HA glycoprotein are responsible for conferring protection against viral infection (12). The antibodies generated against the HA protein during natural infection are primarily directed against the exposed head domain (35). Mutations or recombination events involving the HA and NA genes lead to genetic drift and shift, giving rise to new viruses that are not susceptible to previously acquired immunity by the host. In order to be effective, vaccines have to match the currently circulating viral strains, necessitating the production of new vaccines every season. Therefore, the search for a universal vaccine that provides broader protection and alleviates the need for frequent vaccination is ongoing.A sequence analysis of the HA sequences from various strains and subtypes r...

Purpose: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. Experimental Design: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. Results: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1. Conclusions: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy demonstrating durable responses in multiple tumor types, however only a subset of patients respond, in part due to mechanisms that drive T cell dysfunction. HPK1 is a key inhibitory signaling node associated with T cell exhaustion that is activated in response to T cell receptor engagement (signal 1). HPK1 is thus distinct from inhibitory checkpoint receptors which require surface receptor expression, antigen recognition (signal 1) and costimulatory engagement (signal 2) for activity. HPK1 therefore has potential for broader activity across T cell subsets independent of checkpoint engagement. Selectivity is a critical parameter for the optimal profile of an HPK1 inhibitor because many kinases are positive regulators of T cell signaling and can be antagonistic if inhibited. Utilizing a structure-based drug design approach, we identified compound 1 a highly potent (pSLP76 IC50=55nM) inhibitor of HPK1, which displayed no functional antagonistic effects in human T cells up to 30μM. We attributed this its highly selective nature, possessing >80-fold selectivity against other MAP4K family members, and >25-fold selectivity against all other kinases tested. We demonstrated an inverse correlation between inhibition of pSLP76 and increase in IL-2, IFNg and proliferation in human T cells. We report a novel role for HPK1 in regulating T cell exhaustion in a human ex vivo assay. Compound 1 treatment prevents T cell exhaustion at > 0.1uM and can restore aspects of already exhausted T cells, most notably rescue of cytotoxicity. RNAseq and nuclear localization assays reveal for the first time that HPK1i mechanistically restores AP-1 signaling which enables heterodimerization with NFAT restoring human T cell functionality. We present data demonstrating that our HPK1 inhibitor elevates anti-tumor immunity in multiple syngeneic mouse models including MCA205, CT26 and EMT6. In vitro IC50 correlates with in vivo decrease of pSLP76 in tumors and blood of syngeneic models associated with efficacy. Immunophenotyping from syngeneic tumors treated with compound 1 shows a two-fold increase in CD8+ T cells and most significantly enhanced cytokine production in T cells. In patient NSCLC samples Compound 1 has single agent and combination activity with Durvalumab restoring cytokine production. These results thus reveal the role of AP-1/NFAT signaling in HPK1 biology and highlight the importance of HPK1 in T cell exhaustion in both healthy donor and NSCLC patient tumor T cells, potentially limiting responses to ICB. Citation Format: Deanna A. Mele, Neil Grimster, Gayathri Bommakanti, Jason Shields, Lucas Morrill, Kathryn Giblin, Maryann SanMartin, Yanjun Wang, Minwei Ye, Iswarya KarapaReddy, Kun Song, Theresa Proia, Niresh Hariparsad, Ryan Richards, Nadia Luheshi, Simon T. Barry, Stephen Fawell. First disclosure of a highly potent and selective HPK1 inhibitor that rescues T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3453.