Nonclinical Development of SRK-181: An Anti-Latent TGFß1 Monoclonal Antibody for the Treatment of Locally Advanced or Metastatic Solid Tumors.

Checkpoint inhibitors offer a promising immunotherapy strategy for cancer treatment; however, due to primary or acquired resistance, many patients do not achieve lasting clinical responses. Recently, the transforming growth factor-ß (TGFß) signaling pathway has been identified as a potential target to overcome primary resistance, although the nonselective inhibition of multiple TGFß isoforms has led to dose-limiting cardiotoxicities. SRK-181 is a high-affinity, fully human antibody that selectively binds to latent TGFß1 and inhibits its activation. To support SRK-181 clinical development, we present here a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies showed that SRK-181 has no effect on human platelet function and does not induce cytokine release in human peripheral blood. Four-week toxicology studies with SRK-181 showed that weekly intravenous administration achieved sustained serum exposure and was well tolerated in rats and monkeys, with no treatment-related adverse findings. The no-observed-adverse-effect levels levels were 200 mg/kg in rats and 300 mg/kg in monkeys, the highest doses tested, and provide a nonclinical safety factor of up to 813-fold (based on Cmax) above the phase 1 starting dose of 80 mg every 3 weeks. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that SRK-181 is a selective inhibitor of latent TGFß1 that does not produce the nonclinical toxicities associated with nonselective TGFß inhibition. These data support the initiation and safe conduct of a phase 1 trial with SRK-181 in patients with advanced cancer.

Selective inhibition of TGFß1 activation overcomes primary resistance to checkpoint blockade therapy by altering tumor immune landscape.

Despite breakthroughs achieved with cancer checkpoint blockade therapy (CBT), many patients do not respond to anti-programmed cell death-1 (PD-1) due to primary or acquired resistance. Human tumor profiling and preclinical studies in tumor models have recently uncovered transforming growth factor-ß (TGFß) signaling activity as a potential point of intervention to overcome primary resistance to CBT. However, the development of therapies targeting TGFß signaling has been hindered by dose-limiting cardiotoxicities, possibly due to nonselective inhibition of multiple TGFß isoforms. Analysis of mRNA expression data from The Cancer Genome Atlas revealed that TGFΒ1 is the most prevalent TGFß isoform expressed in many types of human tumors, suggesting that TGFß1 may be a key contributor to primary CBT resistance. To test whether selective TGFß1 inhibition is sufficient to overcome CBT resistance, we generated a high-affinity, fully human antibody, SRK-181, that selectively binds to latent TGFß1 and inhibits its activation. Coadministration of SRK-181-mIgG1 and an anti-PD-1 antibody in mice harboring syngeneic tumors refractory to anti-PD-1 treatment induced profound antitumor responses and survival benefit. Specific targeting of TGFß1 was also effective in tumors expressing more than one TGFß isoform. Combined SRK-181-mIgG1 and anti-PD-1 treatment resulted in increased intratumoral CD8+ T cells and decreased immunosuppressive myeloid cells. No cardiac valvulopathy was observed in a 4-week rat toxicology study with SRK-181, suggesting that selectively blocking TGFß1 activation may avoid dose-limiting toxicities previously observed with pan-TGFß inhibitors. These results establish a rationale for exploring selective TGFß1 inhibition to overcome primary resistance to CBT.

Treatments for the Prevention and Management of Suicide: A Systematic Review.

Background: Suicide is a growing public health problem, with the national rate in the United States increasing by 30% from 2000 to 2016. Purpose: To assess the benefits and harms of nonpharmacologic and pharmacologic interventions to prevent suicide and reduce suicide behaviors in at-risk adults. Data Sources: MEDLINE, EMBASE, PsycINFO, and other databases from November 2011 through May 2018. Study Selection: Systematic reviews (SRs) and randomized controlled trials (RCTs) that assessed nonpharmacologic or pharmacologic therapies for adults at risk for suicide. Data Extraction: One investigator abstracted data and assessed study quality, and a second investigator checked abstractions and assessments for accuracy. Data Synthesis: Eight SRs and 15 RCTs were included. The evidence for psychological interventions suggests that cognitive behavioral therapy (CBT) reduces suicide attempts, suicidal ideation, and hopelessness compared with treatment as usual (TAU). Limited evidence suggests that dialectical behavior therapy (DBT) reduces suicidal ideation compared with wait-list control or crisis planning. The evidence for pharmacologic treatments suggests that ketamine reduces suicidal ideation with minimal adverse events compared with placebo or midazolam. Lithium reduces rates of suicide among patients with unipolar or bipolar mood disorders compared with placebo. However, no differences were observed between lithium and other medications in reducing suicide. Limitation: Qualitative synthesis of new evidence with existing meta-analyses, methodological shortcomings of studies, heterogeneity of nonpharmacologic interventions, and limited evidence for pharmacologic treatments and harms. Conclusion: Both CBT and DBT showed modest benefit in reducing suicidal ideation compared with TAU or wait-list control, and CBT also reduced suicide attempts compared with TAU. Ketamine and lithium reduced the rate of suicide compared with placebo, but there was limited information on harms. Limited data are available to support the efficacy of other nonpharmacologic or pharmacologic interventions. Primary Funding Source: U.S. Department of Veterans Affairs Veterans Health Administration. (PROSPERO: CRD42018104978).

Heterogeneous GM-CSF signaling in macrophages is associated with control of Mycobacterium tuberculosis.

Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF.

Concurrent infection with Mycobacterium tuberculosis confers robust protection against secondary infection in macaques.

For many pathogens, including most targets of effective vaccines, infection elicits an immune response that confers significant protection against reinfection. There has been significant debate as to whether natural Mycobacterium tuberculosis (Mtb) infection confers protection against reinfection. Here we experimentally assessed the protection conferred by concurrent Mtb infection in macaques, a robust experimental model of human tuberculosis (TB), using a combination of serial imaging and Mtb challenge strains differentiated by DNA identifiers. Strikingly, ongoing Mtb infection provided complete protection against establishment of secondary infection in over half of the macaques and allowed near sterilizing bacterial control for those in which a secondary infection was established. By contrast, boosted BCG vaccination reduced granuloma inflammation but had no impact on early granuloma bacterial burden. These findings are evidence of highly effective concomitant mycobacterial immunity in the lung, which may inform TB vaccine design and development.

Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis.

Infection with Mycobacterium tuberculosis causes a spectrum of outcomes; the majority of individuals contain but do not eliminate the infection, while a small subset present with primary active tuberculosis (TB) disease. This variability in infection outcomes is recapitulated at the granuloma level within each host, such that some sites of infection can be fully cleared while others progress. Understanding the spectrum of TB outcomes requires new tools to deconstruct the mechanisms underlying differences in granuloma fate. Here, we use novel genome-encoded barcodes to uniquely tag individual M. tuberculosis bacilli, enabling us to quantitatively track the trajectory of each infecting bacterium in a macaque model of TB. We also introduce a robust bioinformatics pipeline capable of identifying and counting barcode sequences within complex mixtures and at various read depths. By coupling this tagging strategy with serial positron emission tomography coregistered with computed tomography (PET/CT) imaging of lung pathology in macaques, we define a lesional map of M. tuberculosis infection dynamics. We find that there is no significant infection bottleneck, but there are significant constraints on productive bacterial trafficking out of primary granulomas. Our findings validate our barcoding approach and demonstrate its utility in probing lesion-specific biology and dissemination. This novel technology has the potential to greatly enhance our understanding of local dynamics in tuberculosis.IMPORTANCE Classically, M. tuberculosis infection was thought to result in either latent infection or active disease. More recently, the field has recognized that there is a spectrum of M. tuberculosis infection clinical outcomes. Within a single host, this spectrum is recapitulated at the granuloma level, where there can simultaneously be lesional sterilization and poorly contained disease. To better understand the lesional biology of TB infection, we digitally barcoded M. tuberculosis to quantitatively track the fate of each infecting bacterium. By combining this technology with serial PET-CT imaging, we can dynamically track both bacterial populations and granuloma trajectories. We demonstrate that there is little constraint on the bacterial population at the time of infection. However, the granuloma imposes a strong bottleneck on dissemination, and the subset of granulomas at risk of dissemination can be distinguished by physical features.

A Functional Role for Antibodies in Tuberculosis.

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.

A bug's life in the granuloma.

The granuloma is the defining feature of the host response to infection with Mycobacterium tuberculosis (Mtb). Despite knowing of its existence for centuries, much remains unclear regarding the host and bacterial factors that contribute to granuloma formation, heterogeneity of presentation, and the forces at play within. Mtb is highly adapted to life within the granuloma and employs many unique strategies to both create a niche within the host as well as survive the stresses imposed upon it. Adding to the complexity of the granuloma is the vast range of pathology observed, often within the same individual. Here, we explore some of the many ways in which Mtb crafts the immune response to its liking and builds a variety of granuloma features that contribute to its survival. We also consider the multitude of ways that Mtb is adapted to life in the granuloma and how variability in the deployment of these strategies may result in different fates for both the bacterium and the host. It is through better understanding of these complex interactions that we may begin to strategize novel approaches for tuberculosis treatments.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Macrophages clean up: efferocytosis and microbial control.

Phagocytic leukocytes, predominantly macrophages, not only ingest and destroy invading pathogens, but are charged with clearing dead and dying host cells. The process of engulfing apoptotic cells is called efferocytosis and has long been appreciated for its role in the resolution of inflammation. New evidence is emerging that efferocytosis represents a double-edged sword in microbial immunity. Although efferocytosis of influenza and Mycobacterium tuberculosis-infected cells results in pathogen destruction, efferocytosis of Leishmania-infected neutrophils may promote infection. Understanding how macrophages, dendritic cells (DC) and neutrophils process pathogens encased within a dying cell could lead to the development of novel therapeutics that simultaneously suppress inflammation and promote pathogen clearance.

Efferocytosis is an innate antibacterial mechanism.

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.

Critical role for invariant chain in CD1d-mediated selection and maturation of Vα14-invariant NKT cells.

The development and maturation of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated lipid antigen presentation in the thymus and the periphery. Cortical thymocytes mediate positive selection, while professional APCs are involved in thymic negative selection and in terminal maturation of iNKT cells in the periphery. CD1d requires entry in the endosomal pathway to allow antigen acquisition for assembly as lipid/CD1d complexes for display to iNKT cells. This process involves tyrosine-based sorting motifs in the CD1d cytoplasmic tail and invariant chain (Ii) that CD1d associates with in the endoplasmic reticulum. The function of Ii in iNKT cell thymic development and peripheral maturation had not been fully understood. Using mice deficient in Ii and the Ii-processing enzyme cathepsin S (catS), we addressed this question. Ii(-/-) mice but not catS(-/-) mice developed significantly fewer iNKT cells in thymus, that were less mature as measured by CD44 and NK1.1 expression. Ii(-/-) mice but not catS(-/-) mice developed fewer Vß7(+) cells in their iNKT TCR repertoire than WT counterparts, indicative of a change in endogenous glycolipid antigen/CD1d-mediated iNKT cell selection. Finally, using a Mycobacterium tuberculosis infection model in macrophages, we show that iNKT developed in Ii(-/-) but not catS(-/-) mice have defective effector function. Our data support a role for professional APCs expressing Ii, but no role for catS in the thymic development and peripheral terminal maturation of iNKT cells.

Requirement for invariant chain in macrophages for Mycobacterium tuberculosis replication and CD1d antigen presentation.

Mycobacterium tuberculosis is an intracellular bacterium that persists in phagosomes of myeloid cells. M. tuberculosis-encoded factors support pathogen survival and reduce fusion of phagosomes with bactericidal lysosomal compartments. It is, however, not entirely understood if host factors that mediate endosomal fusion affect M. tuberculosis intracellular localization and survival. Neither is it known if endosomal fusion influences induction of host immune reactivity by M. tuberculosis-infected cells. Lysosomal degradation of M. tuberculosis appears to be pivotal for making available lipid substrates for assembly into lipid-CD1d complexes to allow activation of CD1d-restricted invariant natural killer T (iNKT) cells. To clarify the role for endosomal fusion in M. tuberculosis survival and induction of host CD1d-mediated immune defense, we focused our studies on the invariant chain (Ii). Ii regulates endosome docking and fusion and thereby controls endosomal transport. Through direct binding, Ii also directs intracellular transport of the class II major histocompatibility complex and CD1d. Our findings demonstrate that upon infection of Ii-knockout (Ii(-/-)) macrophages, M. tuberculosis is initially retained in early endosomal antigen 1-positive lysosomal-associated membrane protein 1-negative phagosomes, which results in slightly impaired pathogen replication. The absence of Ii did not affect the ability of uninfected and infected macrophages to produce nitric oxide, tumor necrosis factor alpha, or interleukin-12. However, induction of cell surface CD1d was impaired in infected Ii(-/-) macrophages, and CD1d-restricted iNKT cells were unable to suppress bacterial replication when they were cocultured with M. tuberculosis-infected Ii(-/-) macrophages. Thus, while the host factor Ii is not essential for the formation of the M. tuberculosis-containing vacuole, its presence is crucial for iNKT cell recognition of infected macrophages.

Lipids, apoptosis, and cross-presentation: links in the chain of host defense against Mycobacterium tuberculosis.

Eicosanoids regulate whether human and murine macrophages infected with Mycobacterium tuberculosis die by apoptosis or necrosis. The death modality is important since apoptosis is associated with diminished pathogen viability and should be viewed as a form of innate immunity. Apoptotic vesicles derived from infected macrophages are also an important source of bacterial antigens that can be acquired by dendritic cells to prime antigen-specific T cells. This review integrates in vitro and in vivo data on how apoptosis of infected macrophages is linked to development of T cell immunity against M. tuberculosis.