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BackgroundProgrammed cell death protein 1 (PD-1) and CTLA4 combination blockade enhances clinical efficacy in melanoma compared with targeting either checkpoint alone; however, clinical response improvement is coupled with increased risk of developing immune-related adverse events (irAE). Delineating the mechanisms of checkpoint blockade-mediated irAE has been hampered by the lack of animal models that replicate these clinical events.MethodsWe have developed a mouse model of checkpoint blockade-mediated enterocolitis via prolonged administration of an Fc-competent anti-CTLA4 antibody.ResultsSustained treatment with Fc-effector, but not Fc-mutant or Fc-null, anti-CTLA4 antagonist for 7 weeks resulted in enterocolitis. Moreover, combining Fc-null or Fc-mutant CTLA4 antagonists with PD-1 blockade results in potent antitumor combination efficacy indicating that Fc-effector function is not required for combination benefit.ConclusionThese data suggest that using CTLA4 antagonists with no Fc-effector function can mitigate gut inflammation associated with anti-CTLA4 antibody therapy yet retain potent antitumor activity in combination with PD-1 blockade.

Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.

Undesired immune responses against protein therapeutics may adversely affect the pharmacokinetics, efficacy, and safety of the product. The presence of anti-drug-antibodies (ADA) has been the key determinant of immunogenic responses. Here we describe the use of a capillary electrophoresis platform for the identification of ADAs against several experimental camelid VHH biologics (Nanobodies®). Hereafter, we refer to this assay as WESADA. We modified the Wes platform by ProteinSimple to screen serum samples for ADA against covalently linked multi-modular Nanobodies and compared it to standard ADA methodologies. We were able to identify ADA positive samples and determine which individual VHH module in a multivalent Nanobody construct stimulated the predominant ADA response. WESADA requires denaturation of the experimental immobilized drug, which could affect recognition of the immunogenic epitope and alter ADA signal. To address this issue, we demonstrated that signal can be immunodepleted by pre-incubation of serum samples with native Nanobody. This capillary electrophoresis based approach allows for rapid analysis without the need for individually tailored assay optimization or reagent labeling, while consuming small amounts of sample and drug. It also allows for the simultaneous ADA analysis of multiple targets of different molecular size in the same experimental sample. WESADA is not intended to replace traditional ADA assay formats, but it facilitates the expedient immunogenic assessment of a large number of experimental drug candidates in the early developmental space.

IL-33 (IL-1F11) is a recently described member of the IL-1 family of cytokines that stimulates the generation of cells, cytokines, and Igs characteristic of a type 2 immune response. IL-33 mediates signal transduction through ST2, a receptor expressed on Th2 and mast cells. In this study, we demonstrate that IL-33 and ST2 form a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor subunit that is also a member of the IL-1R complex. Additionally, IL-1RAcP is required for IL-33-induced in vivo effects, and IL-33-mediated signal transduction can be inhibited by dominant-negative IL-1RAcP. The implications of this shared usage of IL-1RAcP by IL-1(α and β) and IL-33 are discussed.

Interleukin-12 is a heterodimeric cytokine and an important mediator of the cellular immune response. The recent discovery of the novel cytokine interleukin-23 has led to a re-evaluation of interleukin-12 biology, as both cytokines use a common p40 subunit. This review discusses understanding of what distinguishes these related cytokines and the infection risks associated with targeting these cytokine pathways during treatment of inflammatory diseases.Recent work has shown that interleukin-23 stimulates the development of a distinct subset of effector T cells that produce interleukin-17A. These interleukin-17A-producing cells are critical mediators of the inflammatory response in several mouse models of autoimmunity. Although it is well established that interleukin-12 is a critical mediator of host defense, the role of the interleukin-23/interleukin-17A axis during infections has only recently been evaluated.Interleukin-12 and interleukin-23 have distinct roles in mediating host defense and autoimmune inflammation. Although targeting interleukin-12 and interleukin-23 simultaneously against the common p40 subunit is efficacious in clinical trials for human autoimmune diseases, targeting of interleukin-23 alone or the downstream effector cytokine interleukin-17A may be an effective treatment strategy for organ-specific autoimmune diseases. It is likely that targeting interleukin-23 or interleukin-17A alone, as opposed to targeting interleukin-12 and interleukin-23 together, will reduce the patients' risk of developing treatment-related infections.

Blockade of the checkpoint inhibitor PD-1 has demonstrated remarkable success in the clinic for the treatment of a growing list of different cancers. However, several tumor types are resistant to anti-PD-1 monotherapy. This observation has spurred numerous combination studies to reveal what additional therapeutic interventions may complement anti-PD1 blockade. Recently it has been shown that immunogenic cell death (ICD), induced by radiation and/or chemotherapy, improves T cell responses against different tumor types. ICD is characterized by damage-associated molecular patterns (DAMPs) including surface expression of calreticulin, and release of ATP and HMGB1. Recognition of DAMPs triggers dendritic cell maturation and functions that are critical for tumor antigen-specific T cell activation. Thus therapies that evoke ICD may further augment anti-tumor immunity elicited by anti-PD-1. In mouse syngeneic tumor models, we observed combinatorial anti-tumor activity of anti-PD1 and the cyclin-dependent kinase inhibitor, dinaciclib. We hypothesized that dinaciclib potentiates the effects of anti-PD-1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib express the hallmarks of ICD including HMGB1 and ATP release and surface expression of calreticulin. Dinaciclib treatment also increases tumor cell phagocytosis and induces dendritic cell maturation. Furthermore, mice immunized with dinaciclib-treated tumor cells are resistant to subsequent tumor challenge. Tumors from mice receiving anti-PD1 and dinaciclib have increased T cell infiltration and dendritic cell activation, indicating the overall quality of the immune response generated may be improved with the combo. Taken together, these findings suggest a potential mechanism for the observed synergy between dinaciclib and anti-PD1. Dinaciclib induces immunogenic cell death, converting the tumor cell into an endogenous vaccine and thereby boosting the effects of anti-PD-1. Citation Format: Dewan Mohammed Sakib Hossain, Fernando Ugarte, Anandi Sawant, Mingmei Cai, Venkataraman Sriram, Elaine Pinheiro, Svetlana Sadekova, Alissa Chackerian. Dinaciclib induces immunogenic cell death and enhances anti-PD-1 mediated tumor suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 562.

Host immunity to Mycobacterium tuberculosis is mediated by T cells that recognize and activate infected macrophages to control intracellular bacterial replication. The early appearance of T cells in the lungs of infected mice correlates with greater resistance to infection. However, it is unknown whether the trafficking of T cells to the lung following infection is dependent upon the expression of certain adhesion molecules. To address this question, we infected knockout (KO) mice that have defective expression of CD11a, CD11b, CD18, CD62, CD103, or β7. We found that the integrins CD11a and CD18 are absolutely required for host resistance following infection with aerosolized M. tuberculosis. Although Ag-specific T cells are generated following infection of CD11a KO mice, T cell priming is delayed, T cell trafficking to the lung is impaired, and fewer ESAT6-specific CD4+ T cells are found in the lungs of CD11a KO mice compared with control mice. Thus, LFA-1 (CD11a/CD18) plays an essential role in immunity to M. tuberculosis infection.

Inbred strains of mice exhibit varied patterns of susceptibility following infection with virulent Mycobacterium tuberculosis. Susceptible mice have progressive fulminate disease resulting in their premature death; in contrast, resistant mice are able to control bacterial replication, limit lung injury and survive longer. The use of these mouse strains in experimental infection has allowed the identification of immunological correlates of protective versus unsuccessful host responses to tuberculosis, and the identification of susceptibility loci by combining classical and molecular genetics. These immunological and genetic features that distinguish susceptible and resistant inbred mouse strains may allow us to better understand susceptibility to tuberculous disease in people. A possible benefit would be the delineation of markers of protective immunity for use in vaccine development. This is a review of recent insights into the genetics and immunology of resistance and susceptibility to virulent M. tuberculosis using genetically intact mice.

We report that dissemination ofMycobacterium tuberculosisin the mouse is under host control and precedes the initiation of T-cell immunity. Nine to eleven days after aerosol inoculation,M. tuberculosisdisseminates to the pulmonary lymph nodes (LN), whereM. tuberculosis-specific T cells are detected 2 to 3 days thereafter. This indicates that the initial spread of bacteria occurs via lymphatic drainage and that the acquired T-cell immune response is generated in the draining LN. Dissemination to peripheral sites, such as the spleen and the liver, occurs 11 to 14 days postinfection and is followed by the appearance ofM. tuberculosis-specific T cells in the lung and the spleen. In all cases studied, dissemination to the LN or the spleen preceded activation ofM. tuberculosis-specific T cells in that organ. Interestingly, bacteria disseminate earlier from the lungs of resistant C57BL/6 mice than from the lungs of susceptible C3H mice, and consequently, C57BL/6 mice generate an immune response toM. tuberculosissooner than C3H mice generate an immune response. Thus, instead of spreading infection, early dissemination ofM. tuberculosismay aid in the initiation of an appropriate and timely immune response. We hypothesize that this early initiation of immunity following inoculation withM. tuberculosismay contribute to the superior resistance of C57BL/6 mice.

The human immune system efficiently limits the replication ofMycobacterium tuberculosisin most infected individuals. Only 5 to 10% of infected people develop clinical tuberculosis, a sign of the inability of the immune system to control the infection. We have studied the C3H/HeJ (C3H) and C57BL/6 (B6) inbred mouse strains, which differ in their susceptibility to tuberculosis, in order to ascertain the immunological determinants of a successful immune response againstM. tuberculosisand to establish a system to identify genes that influence susceptibility to tuberculosis. We found that the resistant B6 mice were able to control infection in both the lung and spleen, while susceptible C3H mice were incapable of limiting bacteria growth, especially in the lung, and succumbed to infection within 4 weeks. We determined that the susceptibility of C3H mice was independent of the Toll-like receptor 4 (tlr4) genetic locus and allelic major histocompatibility complex differences. Although the splenic immune responses were similar in the two mouse strains, the local immune responses in the lungs of the infected mice differed greatly. The pulmonary immune response in resistant B6 mice was characterized by an early influx of both CD4+and CD8+lymphocytes that produced gamma interferon (IFN-γ). In contrast, the immune response of C3H mice in the lung was characterized by a delayed and decreased influx of lymphocytes, which produced little IFN-γ. These results suggest an important role for the early appearance of IFN-γ-producing lymphocytes in the lung in resistance to infection withM. tuberculosis.