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). Using insights from the crystal structure of anti-Hu/Cy CD3 antibody ADI-26906 in complex with CD3ε and antibody engineering using a yeast-based platform, we have derived high-affinity CD3 antibody variants with very low polyreactivity and significantly improved biophysical developability. Comparison of these variants with CD3 antibodies in the clinic (as part of bi- or multi-specifics) shows that affinity for CD3 is correlated with polyreactivity. Our engineered CD3 antibodies break this correlation, forming a broad affinity range with no to low polyreactivity. Such antibodies will enable bispecifics with improved pharmacokinetic and safety profiles and suggest engineering solutions that will benefit the large and growing sector of T-cell engagers.

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. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNgamma, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

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. Selective up-regulation of Mcl-1 in PCs by BLyS suggests that this alpha-apoptotic gene product may play an important role in PC survival. Blockade of BLyS, via transmembrane activator and cyclophilin ligand interactor-immunoglobulin treatment, inhibited PC survival in vitro and in vivo. Heightened expression of B cell maturation antigen (BCMA), and lowered expression of transmembrane activator and cyclophilin ligand interactor and BAFF receptor in PCs relative to resting B cells suggests a vital role of BCMA in PC survival. Affirmation of the importance of BCMA in PC survival was provided by studies in BCMA-/- mice in which the survival of long-lived bone marrow PCs was impaired compared with wild-type controls. These findings offer new insights into the molecular basis for the long-term survival of PCs.

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. Neointima formation was also impaired in wild-type mice reconstituted with CD40(-/-) bone marrow. In vitro, the capacity of CD40(-/-) leukocytes to adhere to the endothelium was reduced. Ligated carotid arteries of CD40(-/-) mice showed a smaller total vessel volume and an impaired remodeling capacity, reflected by decreased gelatinolytic/collagenolytic activity. Comparable results were found in mice with defects in CD40-TRAF6 and CD40-TRAF 2/3/5&6 binding, but not in mice with defects in CD40-TRAF2/3&5 binding. Neointima formation and vascular remodeling in CD40-receptor-deficient mice is impaired, due to a decreased inflammatory cell infiltration and matrix-degrading protease activity, with CD40-TRAF6 signaling as the key regulator. This identifies the CD40-TRAF6 axis as a potential therapeutic target in vascular disease.

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. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity.

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. In vivo analysis of antigen-specific CD8(+) T-cell responses revealed that early T-cell expansion was RA-independent; however, late T-cell expansion and clonal accumulation was suppressed strongly in the absence of RA signaling. Our findings indicate that RA function is essential for the survival of tumor-reactive CD8(+) T cells within the TME.

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). VISTA is primarily expressed on hematopoietic cells, and VISTA expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells. A soluble VISTA-Ig fusion protein or VISTA expression on APCs inhibits T cell proliferation and cytokine production in vitro. A VISTA-specific monoclonal antibody interferes with VISTA-induced suppression of T cell responses by VISTA-expressing APCs in vitro. Furthermore, anti-VISTA treatment exacerbates the development of the T cell-mediated autoimmune disease experimental autoimmune encephalomyelitis in mice. Finally, VISTA overexpression on tumor cells interferes with protective antitumor immunity in vivo in mice. These findings show that VISTA, a novel immunoregulatory molecule, has functional activities that are nonredundant with other Ig superfamily members and may play a role in the development of autoimmunity and immune surveillance in cancer.

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. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40-tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe(-/-) mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40-TRAF6, but not CD40-TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40-CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.

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. Patients with measurable metastatic melanoma were treated with intravenous cyclophosphamide (60 mg/kg, days 1 and 2) and fludarabine (25 mg/m(2), day 3 through 7) followed by two 5-day courses of intravenous high-dose bolus IL-2 (600,000 U/kg; days 8 through 12 and 21 through 25). GM-CSF (250 microg/m(2)/d beginning day 8) was given until granulocyte recovery. Lymphocyte recovery profiles were determined by flow cytometric phenotyping at regular intervals, and clinical outcome was assessed by Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS: The trial was stopped at the end of stage 1 with four of 18 objective responses noted. Twelve patients had detailed lymphocyte subcompartments evaluated. After lymphodepletion, we observed an induction of regulatory cells (CD4+ T regulatory cells; CD8+ T suppressor cells) and of T memory cells (CD8+ T central memory cells; T effector memory RA+ cells). Expansion of circulating melanoma-specific CD8(+) cells was observed in one of four HLA-A2-positive patients. CONCLUSION: Chemotherapy-induced lymphodepletion modulates the homeostatic repopulation of the lymphocyte compartment and influences recovering lymphocyte subpopulations. Clinical activity seems similar to standard high-dose aldesleukin alone.

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. Synergistic CD40/TLR activation also induced the migration of activated dendritic cells to lymphatic locations and promoted their capacity to present antigens. Correspondingly, without exogenous antigen, combined CD40/TLR agonists boosted measurable T-cell-mediated antitumor immunity and induced the rejection of otherwise lethal i.p. ovarian carcinomas. Our results highlight the potential of transforming tumor-infiltrating dendritic cells (the most abundant leukocyte subset in the solid ovarian carcinoma microenvironment) from an immunosuppressive to an immunostimulatory cell type. Combined administration of synergistic CD40 and TLR3 agonists could enhance their individual therapeutic effects against ovarian and other lethal epithelial cancers.

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. The Aly mouse expresses a mutant NIK molecule that prohibits the induction of the non-canonical pathway. We show that while MHC class II presentation and in vivo migration of Aly DCs is intact, these cells are unable to cross-prime CD8+ T cells to exogenous Ag. Gene expression array analysis of DCs matured in vivo indicates multiple defects in Ag processing pathways after maturation and provide a global view of the genes that are regulated by the NF-kappaB2 pathway in DCs. These experiments indicate a possible role for NIK in mediating cross-priming of soluble Ag. In addition, our findings explain the profound immune unresponsiveness of the 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. Here, we report that combinatorial use of CD40 and TLR agonists as a cancer vaccine, compared with monotherapy, elicits high frequencies of self-reactive CD8(+) T cells, potent tumor-specific CD8(+) memory, CD8(+) T cells that efficiently infiltrate the tumor-burdened target organ; therapeutic efficacy; heightened ratios of CD8(+) T cells to FoxP3(+) cells at the tumor site; and reduced hepatotoxicity. These findings provide intelligent strategies for the formulation of multifactorial vaccines to achieve maximal efficacy in cancer vaccine trials in humans.

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. Moreover, while lacking germinal center formation, several hallmarks of humoral immune responses including clonal B-cell activation/expansion, antibody isotype switching, and affinity maturation remain normal. This study demonstrates a new functional domain in CD40 that controls critical aspects of B-cell immunity in an in vivo setting.

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.

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. CD40-induced B cell proliferation and early immunoglobulin production occurred even when all TRAF sites were ablated. These studies show that specific CD40-TRAF associations control well defined aspects of humoral immunity. In addition, they define the roles that TRAF-dependent and TRAF-independent pathways play in regulating antigen-driven B cell differentiation.