Fc-enhanced anti-CTLA-4, anti-PD-1, doxorubicin, and ultrasound-mediated BBB opening: A novel combinatorial immunotherapy regimen for gliomas.

BACKGROUND: Glioblastoma is a highly aggressive brain cancer that is resistant to conventional immunotherapy strategies. Botensilimab, an Fc-enhanced anti-CTLA-4 antibody (FcE-aCTLA-4), has shown durable activity in "cold" and immunotherapy-refractory cancers. METHOD: We evaluated the efficacy and immune microenvironment phenotype of a mouse analogue of FcE-aCTLA-4 in treatment-refractory preclinical models of glioblastoma, both as a monotherapy and in combination with doxorubicin delivered via low-intensity pulsed ultrasound and microbubbles (LIPU/MB). Additionally, we studied 4 glioblastoma patients treated with doxorubicin, anti-PD-1 with concomitant LIPU/MB to investigate the novel effect of doxorubicin modulating FcγR expressions in tumor associated macrophages/microglia (TAMs). RESULTS: FcE-aCTLA-4 demonstrated high-affinity binding to FcγRIV, the mouse ortholog of human FcγRIIIA, which was highly expressed in TAMs in human glioblastoma, most robustly at diagnosis. Notably, FcE-aCTLA-4 mediated selective depletion of intra-tumoral regulatory T cells (Tregs) via TAM-mediated phagocytosis, while sparing peripheral Tregs. Doxorubicin, a chemotherapeutic drug with immunomodulatory functions, was found to upregulate FcγRIIIA on TAMs in glioblastoma patients who received doxorubicin and anti-PD-1 with concomitant LIPU/MB. In murine models of immunotherapy-resistant gliomas, a combinatorial regimen of FcE-aCTLA-4, anti-PD-1, and doxorubicin with LIPU/MB, achieved a 90% cure rate, that was associated robust infiltration of activated CD8+ T cells and establishment of immunological memory as evidenced by rejection upon tumor rechallenge. CONCLUSION: Our findings demonstrate that FcE-aCTLA-4 promotes robust immunomodulatory and anti-tumor effects in murine gliomas and is significantly enhanced when combined with anti-PD-1, doxorubicin, and LIPU/MB. We are currently investigating this combinatory strategy in a clinical trial (clinicaltrials.gov NCT05864534).

Molecular mechanism of phosphopeptide neoantigen immunogenicity.

Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL747-755 (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P4 with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization.

Tunable photo-responsive elastic metamaterials.

The metamaterial paradigm has allowed an unprecedented space-time control of various physical fields, including elastic and acoustic waves. Despite the wide variety of metamaterial configurations proposed so far, most of the existing solutions display a frequency response that cannot be tuned, once the structures are fabricated. Few exceptions include systems controlled by electric or magnetic fields, temperature, radio waves and mechanical stimuli, which may often be unpractical for real-world implementations. To overcome this limitation, we introduce here a polymeric 3D-printed elastic metamaterial whose transmission spectrum can be deterministically tuned by a light field. We demonstrate the reversible doubling of the width of an existing frequency band gap upon selective laser illumination. This feature is exploited to provide an elastic-switch functionality with a one-minute lag time, over one hundred cycles. In perspective, light-responsive components can bring substantial improvements to active devices for elastic wave control, such as beam-splitters, switches and filters.

Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication.

Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation.IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

Murine xenograft bioreactors for human immunopeptidome discovery.

The study of peptides presented by MHC class I and class II molecules is limited by the need for relatively large cell numbers, especially when studying post-translationally modified or otherwise rare peptide species. To overcome this problem, we pose the hypothesis that human cells grown as xenografts in immunodeficient mice should produce equivalent immunopeptidomes as cultured cells. Comparing human cell lines grown either in vitro or as murine xenografts, we show that the immunopeptidome is substantially preserved. Numerous features are shared across both sample types, including peptides and proteins featured, length distributions, and HLA-binding motifs. Peptides well-represented in both groups were from more abundant proteins, or those with stronger predicted HLA binding affinities. Samples grown in vivo also recapitulated a similar phospho-immunopeptidome, with common sequences being those found at high copy number on the cell surface. These data indicate that xenografts are indeed a viable methodology for the production of cells for immunopeptidomic discovery.

Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens.

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting T cell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific T cell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory T cell expansion in mice.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication.

The transcription and replication machinery of negative-stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single-domain antibody fragments (VHHs) that protect cells from a lytic infection with vesicular stomatitis virus (VSV) by targeting the viral nucleoprotein N. We define the binding sites for two such VHHs, 1004 and 1307, by X-ray crystallography to better understand their inhibitory properties. We found that VHH 1307 competes with the polymerase cofactor P for binding and thus inhibits replication and mRNA transcription, while binding of VHH 1004 likely only affects genome replication. The functional relevance of these epitopes is confirmed by the isolation of escape mutants able to replicate in the presence of the inhibitory VHHs. The escape mutations allow identification of the binding site of a third VHH that presumably competes with P for binding at another site than 1307. Collectively, these binding sites uncover different features on the N protein surface that may be suitable for antiviral intervention.

Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases.

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer.

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. IMPORTANCE: The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity in vitro The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.

Perspectives on the role of mobility, behavior, and time scales in the spread of diseases.

The dynamics, control, and evolution of communicable and vector-borne diseases are intimately connected to the joint dynamics of epidemiological, behavioral, and mobility processes that operate across multiple spatial, temporal, and organizational scales. The identification of a theoretical explanatory framework that accounts for the pattern regularity exhibited by a large number of host-parasite systems, including those sustained by host-vector epidemiological dynamics, is but one of the challenges facing the coevolving fields of computational, evolutionary, and theoretical epidemiology. Host-parasite epidemiological patterns, including epidemic outbreaks and endemic recurrent dynamics, are characteristic to well-identified regions of the world; the result of processes and constraints such as strain competition, host and vector mobility, and population structure operating over multiple scales in response to recurrent disturbances (like El Niño) and climatological and environmental perturbations over thousands of years. It is therefore important to identify and quantify the processes responsible for observed epidemiological macroscopic patterns: the result of individual interactions in changing social and ecological landscapes. In this perspective, we touch on some of the issues calling for the identification of an encompassing theoretical explanatory framework by identifying some of the limitations of existing theory, in the context of particular epidemiological systems. Fostering the reenergizing of research that aims at disentangling the role of epidemiological and socioeconomic forces on disease dynamics, better understood as complex adaptive systems, is a key aim of this perspective.

Phenotypic lentivirus screens to identify functional single domain antibodies.

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway.

Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy.

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

The social benefits of private infectious disease-risk mitigation.

Does society benefit from private measures to mitigate infectious disease risks? Since mitigation reduces both peak prevalence and the number of people who fall ill, the answer might appear to be yes. But mitigation also prolongs epidemics and therefore the time susceptible people engage in activities to avoid infection. These avoidance activities come at a cost-in lost production or consumption, for example. Whether private mitigation yields net social benefits depends on the social weight given to the costs of illness and illness avoidance, now and into the future. We show that, for a large class of infectious diseases, private risk mitigation is socially beneficial. However, in cases where society discounts the future at either very low or very high rates relative to private individuals, or where it places a low weight on the private cost of illness, the social cost of illness under proportionate mixing (doing nothing) may be lower than the social cost of illness under preferential mixing (avoiding infectious individuals). That is, under some circumstances, society would prefer shorter, more intense epidemics without avoidance costs over longer, less intense epidemics with avoidance costs. A sobering (although not surprising) implication of this is that poorer societies should be expected to promote less private disease-risk mitigation than richer societies.

Resource consumption, sustainability, and cancer.

Preserving a system's viability in the presence of diversity erosion is critical if the goal is to sustainably support biodiversity. Reduction in population heterogeneity, whether inter- or intraspecies, may increase population fragility, either decreasing its ability to adapt effectively to environmental changes or facilitating the survival and success of ordinarily rare phenotypes. The latter may result in over-representation of individuals who may participate in resource utilization patterns that can lead to over-exploitation, exhaustion, and, ultimately, collapse of both the resource and the population that depends on it. Here, we aim to identify regimes that can signal whether a consumer-resource system is capable of supporting viable degrees of heterogeneity. The framework used here is an expansion of a previously introduced consumer-resource type system of a population of individuals classified by their resource consumption. Application of the Reduction Theorem to the system enables us to evaluate the health of the system through tracking both the mean value of the parameter of resource (over)consumption, and the population variance, as both change over time. The article concludes with a discussion that highlights applicability of the proposed system to investigation of systems that are affected by particularly devastating overly adapted populations, namely cancerous cells. Potential intervention approaches for system management are discussed in the context of cancer therapies.

Merging economics and epidemiology to improve the prediction and management of infectious disease.

Mathematical epidemiology, one of the oldest and richest areas in mathematical biology, has significantly enhanced our understanding of how pathogens emerge, evolve, and spread. Classical epidemiological models, the standard for predicting and managing the spread of infectious disease, assume that contacts between susceptible and infectious individuals depend on their relative frequency in the population. The behavioral factors that underpin contact rates are not generally addressed. There is, however, an emerging a class of models that addresses the feedbacks between infectious disease dynamics and the behavioral decisions driving host contact. Referred to as "economic epidemiology" or "epidemiological economics," the approach explores the determinants of decisions about the number and type of contacts made by individuals, using insights and methods from economics. We show how the approach has the potential both to improve predictions of the course of infectious disease, and to support development of novel approaches to infectious disease management.

Disease risk mitigation: the equivalence of two selective mixing strategies on aggregate contact patterns and resulting epidemic spread.

The personal choices affecting the transmission of infectious diseases include the number of contacts an individual makes, and the risk-characteristics of those contacts. We consider whether these different choices have distinct implications for the course of an epidemic. We also consider whether choosing contact mitigation (how much to mix) and affinity mitigation (with whom to mix) strategies together has different epidemiological effects than choosing each separately. We use a set of differential equation compartmental models of the spread of disease, coupled with a model of selective mixing. We assess the consequences of varying contact or affinity mitigation as a response to disease risk. We do this by comparing disease incidence and dynamics under varying contact volume, contact type, and both combined across several different disease models. Specifically, we construct a change of variables that allows one to transition from contact mitigation to affinity mitigation, and vice versa. In the absence of asymptomatic infection we find no difference in the epidemiological impacts of the two forms of disease risk mitigation. Furthermore, since models that include both mitigation strategies are underdetermined, varying both results in no outcome that could not be reached by choosing either separately. Which strategy is actually chosen then depends not on their epidemiological consequences, but on the relative cost of reducing contact volume versus altering contact type. Although there is no fundamental epidemiological difference between the two forms of mitigation, the social cost of alternative strategies can be very different. From a social perspective, therefore, whether one strategy should be promoted over another depends on economic not epidemiological factors.

Sensitivity of the polymerase of vesicular stomatitis virus to 2' substitutions in the template and nucleotide triphosphate during initiation and elongation.

The RNA synthesis machinery of non-segmented negative-sense RNA viruses comprises a ribonucleoprotein complex of the genomic RNA coated by a nucleocapsid protein (N) and associated with polymerase. Work with vesicular stomatitis virus (VSV), a prototype, supports a model of RNA synthesis whereby N is displaced from the template to allow the catalytic subunit of the polymerase, the large protein (L) to gain access to the RNA. Consistent with that model, purified L can copy synthetic RNA that contains requisite promoter sequences. Full processivity of L requires its phosphoprotein cofactor and the template-associated N. Here we demonstrate the importance of the 2' position of the RNA template and the substrate nucleotide triphosphates during initiation and elongation by L. The VSV polymerase can initiate on both DNA and RNA and can incorporate dNTPs. During elongation, the polymerase is sensitive to 2' modifications, although dNTPs can be incorporated, and mixed DNA-RNA templates can function. Modifications to the 2' position of the NTP, including 2',3'-ddCTP, arabinose-CTP, and 2'-O-methyl-CTP, inhibit polymerase, whereas 2'-amino-CTP is incorporated. The inhibitory effects of the NTPs were more pronounced on authentic N-RNA with the exception of dGTP, which is incorporated. This work underscores the sensitivity of the VSV polymerase to nucleotide modifications during initiation and elongation and highlights the importance of the 2'-hydroxyl of both template and substrate NTP. Moreover, this study demonstrates a critical role of the template-associated N protein in the architecture of the RNA-dependent RNA polymerase domain of L.

The polymerase of negative-stranded RNA viruses.

Negative-sense (NS) RNA viruses deliver into cells a mega-dalton RNA-protein complex competent for transcription. Within this complex, the RNA is protected in a nucleocapsid protein (NP) sheath which the viral polymerase negotiates during RNA synthesis. The NP-RNA templates come as nonsegmented (NNS) or segmented (SNS), necessitating distinct strategies for transcription by their polymerases. Atomic-level understanding of the NP-RNA of both NNS and SNS RNA viruses show that the RNA must be transiently dissociated from NP during RNA synthesis. Here we summarize and compare the polymerases of NNS and SNS RNA viruses, and the current structural data on the polymerases. Those comparisons inform us on the evolution of related RNA synthesis machines which use two distinct mechanisms for mRNA cap formation.