Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity.

Bispecific antibodies continue to represent a growth area for antibody therapeutics, with roughly a third of molecules in clinical development being T-cell engagers that use an anti-CD3 binding arm. CD3 antibodies possessing cross-reactivity with cynomolgus monkey typically recognize a highly electronegative linear epitope at the extreme N-terminus of CD3 epsilon (CD3ε). Such antibodies have high isoelectric points and display problematic polyreactivity (correlated with poor pharmacokinetics for monospecific antibodies).

Tryptophan hydroxylase-1 regulates immune tolerance and inflammation.

Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively.

A retinoic acid--rich tumor microenvironment provides clonal survival cues for tumor-specific CD8(+) T cells.

While vitamin A has been implicated in host resistance to infectious disease, little is known about the role of vitamin A and its active metabolite, retinoic acid (RA) in host defenses against cancer. Here, we show that local RA production within the tumor microenvironment (TME) is increased up to 5-fold as compared with naïve surrounding tissue, with a commensurate increase in RA signaling to regionally infiltrating tumor-reactive T cells. Conditional disruption of RA signaling in CD8(+) T cells using a dominant negative retinoic acid receptor α (dnRARα) established that RA signaling is required for tumor-specific CD8(+) T-cell expansion/accumulation and protective antitumor immunity.

VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses.

The immunoglobulin (Ig) superfamily consists of many critical immune regulators, including the B7 family ligands and receptors. In this study, we identify a novel and structurally distinct Ig superfamily inhibitory ligand, whose extracellular domain bears homology to the B7 family ligand PD-L1. This molecule is designated V-domain Ig suppressor of T cell activation (VISTA).

Cytokine working group study of lymphodepleting chemotherapy, interleukin-2, and granulocyte-macrophage colony-stimulating factor in patients with metastatic melanoma: clinical outcomes and peripheral-blood cell recovery.

Purpose: Recovery of lymphocyte populations after lymphocyte depletion is implicated in therapeutic immune pathways in animal models and in patients with cancer. We sought to evaluate the effects of chemotherapy-induced lymphodepletion followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) and high-dose interleukin-2 (IL-2) therapy on clinical response and the recovery of lymphocyte subcompartments in patients with metastatic melanoma.

Patients And Methods: This was a two-stage phase II trial design.

Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile.

The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)-deficient (Apoe(-/-)) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis.

In situ stimulation of CD40 and Toll-like receptor 3 transforms ovarian cancer-infiltrating dendritic cells from immunosuppressive to immunostimulatory cells.

Boosting therapeutically relevant immunity against lethal epithelial tumors may require targeting tumor-induced immunosuppression on an individualized basis. Here, we show that, in the ovarian carcinoma microenvironment, CD11c(+)MHC-II(+) dendritic cells spontaneously engulf tumor materials but, rather than enhancing antitumor immunity, suppress T-cell function. In situ costimulation of CD40 and Toll-like receptor (TLR) 3 on tumor-infiltrating dendritic cells decreased their L-arginase activity, enhanced their production of type I IFN and interleukin-12 (p70), augmented their capacity to process antigens, and up-regulated costimulatory molecules in vivo in mice and in vitro in human dissociated tumors.

Dendritic cells require the NF-kappaB2 pathway for cross-presentation of soluble antigens.

NF-kappaB-inducing kinase (NIK) is responsible for activation of the non-canonical p100 processing pathway of NF-kappaB activation. This kinase has been shown to be critical for activation of this pathway after signaling through several TNF family members including CD40. The functional importance of this pathway in CD40 and TLR-induced dendritic cell (DC) differentiation was studied in vivo in the alymphoplasia (Aly) mouse.

Enhanced efficacy and reduced toxicity of multifactorial adjuvants compared with unitary adjuvants as cancer vaccines.

Identification of Toll-like receptors (TLRs) and their ligands, and tumor necrosis factor-tumor necrosis factor receptor (TNF-TNFR) pairs have provided the first logical, hypothesis-based strategies to molecularly concoct adjuvants to elicit potent cell-mediated immunity via activation of innate and adaptive immunity. However, isolated activation of one immune pathway in the absence of others can be toxic, ineffective, and detrimental to long-term, protective immunity. Effective engineered vaccines must include agents that trigger multiple immunologic pathways.

The CD40-TRAF6 axis is the key regulator of the CD40/CD40L system in neointima formation and arterial remodeling.

We investigated the role of CD40 and CD40L in neointima formation and identified the downstream CD40-signaling intermediates (tumor necrosis factor [TNF]-receptor associated factors [TRAF]) involved. Neointima formation was induced in wild-type, CD40(-/-), CD40L(-/-), and in CD40(-/-) mice that contained a CD40 transgene with or without mutations at the CD40-TRAF2,3&5, TRAF6, or TRAF2,3,5&6 binding sites. Compared with wild-type mice, CD40(-/-) mice showed a significant decrease in neointima formation with increased collagen deposition and decreased inflammatory cell infiltration.

The in vivo function of a noncanonical TRAF2-binding domain in the C-terminus of CD40 in driving B-cell growth and differentiation.

The recruitment of tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 to the CD40 cytoplasmic tail upon CD40 trimerization results in downstream signaling events that ultimately lead to CD40-dependent, thymus-dependent (TD) humoral immune responses. Previously, we have shown signaling through the C-terminal tail of CD40 in the absence of canonical TRAF-binding sites is capable of signaling through an alternative TRAF2-binding site. Here, we demonstrate that B cells from mice harboring CD40 with only the C-terminal tail can activate both canonical and noncanonical NFkappaB signaling pathways.

CD40-associated TRAF 6 signaling is required for disease induction in a retrovirus-induced murine immunodeficiency.

LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.

Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN.

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10-20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)gamma production and an enhanced secondary response to antigenic challenge.

BCMA is essential for the survival of long-lived bone marrow plasma cells.

Long-lived humoral immunity is manifested by the ability of bone marrow plasma cells (PCs) to survive for extended periods of time. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes. We show that BLyS can sustain PC survival in vitro, and this survival can be further enhanced by interleukin 6.

The CD40-TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells.

Affinity maturation of the immune response and the generation of long-lived bone marrow (BM) plasma cells are hallmarks of CD40-dependent, thymus-dependent (TD) humoral immunity. Through disruption of the tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-binding site within the CD40 cytoplasmic domain, we selectively ablated affinity maturation and the generation of plasma cells after immunization. Mutagenesis of both the TRAF6 and TRAF2-TRAF3 sites was essential for arresting germinal center formation in response to immunization.

Regulation of G(1) cyclin-dependent kinases in the liver: role of nuclear localization and p27 sequestration.

Recent studies suggest that cyclin D1 mediates progression of hepatocytes through G(1) phase of the cell cycle. The present study further examines the regulation of cyclin D1-dependent kinase activity and the interplay between cyclin D1 and other G(1) phase regulatory proteins during liver regeneration. After 70% partial hepatectomy in rats, there was upregulation of kinase activity associated with cyclins (A, D1, D3, and E), cyclin-dependent kinases (Cdk2 and Cdk4), and Cdk-inhibitory proteins (p27, p107, and p130).

Dendritic cell maturation and subsequent enhanced T-cell stimulation induced with the novel synthetic immune response modifier R-848.

Agents that enhance dendritic cell maturation can enhance T-cell activation and therefore may improve the efficiency of vaccines or improve cellular immunotherapy. Previously, we demonstrated that a novel low-molecular-weight synthetic immune response modifier, R-848, induces IL-12 and IFN-alpha secretion from monocytes and macrophages. Here we report that R-848 induces the maturation of human monocyte-derived dendritic cells.

Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod.

Cytokines produced by antigen-presenting cells are known to affect the development and cytokine profile of T cells. The immune response modifiers imiquimod and R-848 were previously shown to stimulate human and mouse cultures to secrete interferon-alpha. Results from the present study demonstrate that R-848 and imiquimod are capable of inducing interleukin-12 and interferon-gamma in mouse and human cell cultures.

Involvement of p21 and p27 in the regulation of CDK activity and cell cycle progression in the regenerating liver.

In tissue culture systems, p21 and p27 inhibit cyclin-dependent kinase (CDK) activity and cell cycle progression in response to numerous stimuli, but little is known about their involvement in cell growth in vivo. We examined the modulation of CDK activity by these proteins after 70% partial hepatectomy (PH), an in vivo model of synchronous hepatocyte cell cycle progression. After PH in BALB/c mice, p21 was induced during the prereplicative (G1) phase and was maximally expressed after peak hepatocyte DNA synthesis.