Projected Dose Optimization of Amino- and Hydroxypyrrolidine Purine PI3Kδ Immunomodulators.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells.

Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.

Targeting antigens to CD180 rapidly induces antigen-specific IgG, affinity maturation, and immunological memory.

Antigen (Ag) targeting is an efficient way to induce immune responses. Ag is usually coupled to an antibody (Ab) specific for a receptor expressed on dendritic cells (DCs), and then the Ag-anti-receptor is inoculated with an adjuvant. Here we report that targeting Ag to a receptor expressed on both B cells and DCs, the TLR orphan receptor CD180, in the absence of adjuvant rapidly induced IgG responses that were stronger than those induced by Ag in alum. Ag conjugated to anti-CD180 (Ag-αCD180) induced affinity maturation and Ab responses that were partially T cell independent, as Ag-specific IgGs were generated in CD40- and T cell-deficient mice. After preimmunization with Ag-αCD180 and boosting with soluble Ag, both WT and CD40 knockout (KO) mice rapidly produced Ag-specific IgG-forming cells, demonstrating that Ag-anti-CD180 induces immunological memory. The potent adjuvant effect of Ag-αCD180 required Ag to be coupled to anti-CD180 and the responsive B cells to express both CD180 and an Ag-specific B cell receptor. Surprisingly, CD180 Ag targeting also induced IgG Abs in BAFF-R KO mice lacking mature B cells and in mice deficient in interferon signaling. Targeting Ag to CD180 may be useful for therapeutic vaccination and for vaccinating the immune compromised.

CD28-B7 interaction modulates short- and long-lived plasma cell function.

The interaction of CD28, which is constitutively expressed on T cells, with B7.1/B7.2 expressed on APCs is critical for T cell activation. CD28 is also expressed on murine and human plasma cells but its function on these cells remains unclear. There are two types of plasma cells: short-lived ones that appear in the secondary lymphoid tissue shortly after Ag exposure, and long-lived plasma cells that mainly reside in the bone marrow. We demonstrate that CD28-deficient murine short- and long-lived plasma cells produce significantly higher levels of Abs than do their wild-type counterparts. This was owing to both increased frequencies of plasma cells as well as increased Ab production per plasma cell. Plasma cells also express the ligand for CD28, B7.1, and B7.2. Surprisingly, deficiency of B7.1 and B7.2 in B cells also led to higher Ab levels, analogous to Cd28(-/-) plasma cells. Collectively, our results suggest that the CD28-B7 interaction operates as a key modulator of plasma cell function.

The RIG-I-like receptor LGP2 controls CD8(+) T cell survival and fitness.

The RIG-I-like receptors (RLRs) signal innate immune defenses upon RNA virus infection, but their roles in adaptive immunity have not been clearly defined. Here, we showed that the RLR LGP2 was not essential for induction of innate immune defenses, but rather was required for controlling antigen-specific CD8(+) T cell survival and fitness during peripheral T cell-number expansion in response to virus infection. Adoptive transfer and biochemical studies demonstrated that T cell-receptor signaling induced LGP2 expression wherein LGP2 operated to regulate death-receptor signaling and imparted sensitivity to CD95-mediated cell death. Thus, LGP2 promotes an essential prosurvival signal in response to antigen stimulation to confer CD8(+) T cell-number expansion and effector functions against divergent RNA viruses, including West Nile virus and lymphocytic choriomeningitis virus.

Immune complex-mediated enhancement of secondary antibody responses.

Immunologic memory is a hallmark of the vertebrate immune system. The first antigenic exposure leads to a slow and modest immune response, whereas repeated exposure, even many years later, leads to a rapid and exaggerated response that is two to three orders of magnitude greater than the primary. In the case of humoral immunity, the increased efficacy of recall responses is due to the production of amplified levels of Ag-specific Ab, as well as the accelerated kinetics of their production. Current thinking suggests that this is due to selective activation of long-lived, Ag-specific memory B cells. A downside of restricting secondary responses solely to memory cells is that the repertoire of the memory B cell pool remains static while pathogens continue to evolve. In this study, we propose that during secondary responses, naive Ag-specific B cells participate alongside memory cells. We show that immune complexes formed in vivo between the Ag and pre-existing Abs from the primary response activate these naive B cells, inducing them to respond with accelerated kinetics and increased magnitude. Thus, the continued recruitment of new B cell clones after each antigenic exposure enables the immune system to stay abreast of rapidly changing pathogens.