A bispecific anti-PD-1 and PD-L1 antibody induces PD-1 cleavage and provides enhanced anti-tumor activity.

Combinatorial strategies, such as targeting different immune checkpoint receptors, hold promise to increase the breadth and duration of the response to cancer therapy. Here we describe the preclinical evaluation of CTX-8371, a protein construct which combines PD-1 and PD-L1 targeting in one bispecific, tetravalent antibody. CTX-8371 matched or surpassed the activity of anti-PD-1 and PD-L1 benchmark antibodies in several in vitro T cell activation assays and outperformed clinically approved benchmarks in the subcutaneous MC38 colon and the B16F10 lung metastasis mouse tumor models. Investigation into the mechanism of action revealed that CTX-8371 co-engagement of PD-1 and PD-L1 induced the proteolytic cleavage and loss of cell surface PD-1, which is a novel and non-redundant mechanism that adds to the PD-1/PD-L1 signaling axis blockade. The combination of CTX-8371 and an agonistic anti-CD137 antibody further increased the anti-tumor efficacy with long-lasting curative therapeutic effect. In summary, CTX-8371 is a novel checkpoint inhibitor that might provide greater clinical benefit compared to current anti-PD-1 and PD-L1 antibodies, especially when combined with agents with orthogonal mechanisms of action, such as agonistic anti-CD137 antibodies.

The preclinical efficacy, selectivity and pharmacologic profile of MK-5932, an insulin-sparing selective glucocorticoid receptor modulator.

Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.

Lipid nanoparticle purification by spin centrifugation-dialysis (SCD): a facile and high-throughput approach for small scale preparation of siRNA-lipid complexes.

This paper describes the use of spin centrifugation-dialysis (SCD) for small-scale concentration/purification of siRNA-lipid complexes designed for use as therapeutic agents for gene silencing. SCD consists of a two-step method for concentration, filtration and buffer exchange of lipid nanoparticles (LNP) to provide a homogeneous preparation suitable for injection. Here, we compare SCD with the more traditionally used tangential flow filtration (TFF), and demonstrate the physicochemical and biological comparability of LNPs produced with both methods. TFF is a highly scalable method used in both developmental and production applications, but is limited in terms of miniaturization. In contrast to TFF, SCD is faster, less expensive, and requires less oversight for assembling LNPs for small-scale applications, such as target screening both in vitro and in vivo. The finding that SCD is a viable method for filtering LNPs in a manner similar to TFF, producing particles with comparable properties and biological activity, is significant given the complexity and sensitivity of LNPs to processing conditions.

Choice of fixative is crucial to successful immunohistochemical detection of phosphoproteins in paraffin-embedded tumor tissues.

Protein phosphorylation is frequently used as an indicator of cellular signaling activity. Elevated phosphorylation of tyrosine kinase receptors plays an important role in cancer pathogenesis. However, phosphoproteins are usually poorly preserved in clinical tissue samples that are routinely fixed in 10% formalin. Nonetheless, in oncology clinical trials, use of phosphoproteins as biomarkers has been considered to be of great value in evaluating the effectiveness of a given drug candidate. Therefore, it is worthy of investigating whether alternative fixatives would improve the preservation of phosphoproteins in tissue. We compared the IHC staining of a number of phosphoproteins in xenograft and human surgical tumor tissues fixed in three different fixatives: 10% formalin, 4% paraformaldehyde (PFA), and Streck's tissue fixative (STF). We found that STF significantly enhanced the staining intensity of phosphoproteins compared with 10% formalin or 4% PFA. STF fixative also showed superiority of preservation of phosphoproteins in human surgical samples. Our results indicate that the choice of fixative could significantly affect the usability of clinical tissue samples for evaluating phosphoprotein by IHC.

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model.

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

Effect of vaccine delivery system on the induction of HPV16L1-specific humoral and cell-mediated immune responses in immunized rhesus macaques.

There have been numerous studies to assess the immunogenicity of candidate therapeutic and prophylactic vaccines for human papillomavirus (HPV), but few of them have directly compared different vaccines in an immunologically relevant animal system. In the present study, several vaccine delivery systems (VLPs, chimeric VLPs, plasmid DNA, and a replication incompetent adenoviral vector) expressing HPV16L1 were evaluated for their ability to induce HPV16L1 VLP-specific humoral immune responses, including neutralizing antibodies, and cell-mediated immune responses in rhesus macaques. Monkeys immunized with HPV16L1 VLPs mounted a potent humoral response with strongly neutralizing antibodies and a strong L1-specific Th2 response as measured by IL-4 production by CD4+ T cells. Monkeys immunized with plasmid DNA or an adenoviral vector expressing HPV16L1 showed strong Th1/Tc1 responses as measured by IFN-gamma production by CD4+ and/or CD8+ T cells and potent humoral responses, but only weakly neutralizing antibodies. These data demonstrate that the nature of the immune response against HPV16L1 is dramatically different when it is introduced via different delivery systems. Additionally, these findings support the notion that an HPV16L1 VLP-based vaccine will induce the strongly neutralizing antibodies necessary for effective prophylaxis.

Correlation of tissue distribution, developmental phenotype, and intestinal homing receptor expression of antigen-specific B cells during the murine anti-rotavirus immune response.

The intestinal homing receptor, alpha(4)beta(7), helps target lymphocytes to Peyer's patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing alpha(4)beta(7). In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD(-)B220(low)alpha(4)beta(7)(-)and alpha(4)beta(7)(+) cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD(-)B220(high)alpha(4)beta(7)(-)cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD(-)B220(low)alpha(4)beta(7)(+) cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express alpha(4)beta(7). Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that alpha(4)beta(7) is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues.