APX005M, a CD40 agonist antibody with unique epitope specificity and Fc receptor binding profile for optimal therapeutic application.

Targeting CD40 with agonist antibodies is a promising approach to cancer immunotherapy. CD40 acts as a master regulator of immunity by mobilizing multiple arms of the immune system to initiate highly effective CD8 + T-cell-mediated responses against foreign pathogens and tumors. The clinical development of CD40 agonist antibodies requires careful optimization of the antibody to maximize therapeutic efficacy while minimizing adverse effects. Both epitope specificity and isotype are critical for CD40 agonist antibody mechanism of action and potency. We developed a novel antibody, APX005M, which binds with high affinity to the CD40 ligand-binding site on CD40 and is optimized for selective interaction with Fcγ receptors to enhance agonistic potency while limiting less desirable Fc-effector functions like antibody-dependent cellular cytotoxicity of CD40-expressing immune cells. APX005M is a highly potent inducer of innate and adaptive immune effector responses and represents a promising CD40 agonist antibody for induction of an effective anti-tumor immune response with a favorable safety profile.

Plasmacytoid dendritic cell dichotomy: identification of IFN-α producing cells as a phenotypically and functionally distinct subset.

Plasmacytoid dendritic cells (pDC) produce large amounts of type I IFN in response to invading pathogens, but can also suppress immune responses and promote tolerance. In this study, we show that in mice, these functions are attributable to two distinct pDC subsets, one of which gives rise to the other. CD9(pos)Siglec-H(low) pDC secrete IFN-α when stimulated with TLR agonists, induce CTLs, and promote protective antitumor immunity. By contrast, CD9(neg)Siglec-H(high) pDC secrete negligible amounts of IFN-α, induce Foxp3(+) CD4(+) T cells, and fail to promote antitumor immunity. Although newly formed pDC in the bone marrow are CD9(pos) and are capable of producing IFN-α, after these cells traffic to peripheral tissues, they lose CD9 expression and the ability to produce IFN-α. We propose that newly generated pDC mobilized from the bone marrow, rather than tissue-resident pDC, are the major source of IFN-α in infected hosts.

Plasmacytoid dendritic cells take up opsonized antigen leading to CD4+ and CD8+ T cell activation in vivo.

Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.

Influenza A virus elevates active cathepsin B in primary murine DC.

Dendritic cells (DCs) act as a first-line recognition system for invading pathogens, such as influenza A. The interaction of DC with influenza A virus results in DC activation via endosomal Toll-like receptors and also leads to presentation of viral peptides on MHC class II molecules. Prior work demonstrated that influenza A virus (A/HKx31; H3N2) infection of BALB/c mice activates lung DCs for antigen presentation, and that the enhanced function of these cells persists long after viral clearance and resolution of the virus-induced inflammatory response. Whether influenza A virus has acute or longer-lasting effects on the endo/lysosomal antigen-processing machinery of DCs has not been studied. Here, we show that antigen presentation from intact protein antigen, but not peptide presentation, results in increased T cell stimulation by influenza-exposed lung DCs, suggesting increased antigen processing/loading in these DCs. We find that cathepsin (Cat) B levels and activity are substantially up-regulated in murine lung DCs, harvested 30 days after A/HKx31 infection. CatB levels and activity are also increased in murine splenic and bone marrow-derived DCs, following short-term in vitro exposure to UV-inactivated influenza A virus. Modest effects on CatX are also seen during in vivo and in vitro exposure to influenza A virus. Using a cell permeable Cat inhibitor, we show Cats in influenza-exposed DCs to be functional and required for generation of a T cell epitope from intact ovalbumin. Our findings indicate that influenza A virus affects the MHC class II antigen-processing pathway, an essential pathway for CD4(+) T cell activation.

Dendritic cells exposed to herpes simplex virus in vivo do not produce IFN-alpha after rechallenge with virus in vitro and exhibit decreased T cell alloreactivity.

Plasmacytoid dendritic cells (DC) are known to produce large amounts of IFN-alpha when stimulated with virus in vivo and in vitro. Immunohistological staining of spleens from mice taken at different times after HSV infection revealed an early infiltration of plasmacytoid DC whereas both the myeloid DC and lymphoid-related DC had different kinetics. Upon rechallenge with virus in vitro, total splenic DCs from viral-infected mice were unable to produce IFN-alpha when compared with DC from mice that received an initial in vivo injection with PBS. Furthermore, DC from mice that were infected with increasing doses of HSV expressed high levels of accessory and activation molecules compared with control mice. However, when cultured in vitro together with allogeneic T cells, DC from mice that had been exposed to the highest viral titers in vivo induced the lowest levels of T cell proliferation. DC exposed to PBS in vivo promoted a Th1 response upon coculture with CD4(+) T cells whereas T cells cultured with DC exposed to increasing viral titers in vivo resulted in a gradually decreased Th1 response. The data suggest HSV induces DC maturation and at higher titers, exhaustion, diminishing T cell proliferation, and IFN-gamma secretion.

The multifaceted murine plasmacytoid dendritic cell.

Plasmacytoid dendritic cells (PDCs) or natural interferon-producing cells, function as the body's innate defense against viral infections. As discussed here, they may play additional roles in response to bacterial pathogens and may have the capacity to induce different type of T-cell responses depending on what signals they receive. The discovery of murine PDCs will allow for the design of models to study viral immunobiology in vivo and to determine their function in various diseases that involve plasmacytoid dendritic cells, such as selected leukemias, lymphomas, allergies, different autoimmune conditions, and their possible role in inducing and maintaining tolerance.

Progenipoietin-generated dendritic cells exhibit anti-tumor efficacy in a therapeutic murine tumor model.

Progenipoietin (ProGP-4) is a chimeric molecule, exhibiting both Flt-3 and granulocyte-colony stimulating factor (G-CSF) receptor agonist activities. Subcutaneous administration of ProGP-4 to BALB/c mice at a dose of 40-100 microg/day for up to 12 consecutive days induces both CD11c(+) dendritic cells (DCs) and CD11c(-)/CD11b(+) granulocytes in spleen, blood and lymph nodes of treated animals. Peak numbers of all cell populations were observed on day 7 of treatment, with CD11c(+) DCs representing approximately 8% of total splenocytes at that time. Approximately 40-50% of these CD11c(+) cells were also able to endocytose and process the exogenous fluorescent antigen DQ-BSA. As a test of their therapeutic utility, freshly prepared CD11c(+) DCs were pulsed with a defined tumor-associated peptide epitope (murine p53(232-240)) and injected as a vaccine into BALB/c mice bearing day 7 established CMS4 sarcomas. Similarly prepared DCs were injected again 1 week later. Based on our results, we conclude that (i) both peptide-pulsed CD11c(+) DCs (harvested directly from ProGP-4 treated mice) and pulsed bone marrow-derived DCs effectively slow the growth of or mediate the regression (in 25 of 89 [28%] cases) of CMS4 tumors, and (ii) nonpulsed DCs mediated minimal or no therapeutic effect. These data support the ability of ProGP-4 to enhance the peripheral frequencies of DCs that exhibit therapeutic efficacy when applied as a vaccine to treat tumor-bearing animals.