Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity.

Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies.

The Effect of ASP2409, a Novel CD86-Selective Variant of CTLA4-Ig, on Renal Allograft Rejection in Nonhuman Primates.

Background: Blockade of CD28-mediated T cell costimulation by a modified cytotoxic T lymphocyte-associated antigen 4 (CTLA4-Ig), belatacept, is a clinically effective immunosuppressive therapy for the prevention of renal allograft rejection. Use of belatacept-based calcineurin inhibitor-free immunosuppression, however, has demonstrated an increased frequency of cellular rejection episodes and immunosuppression-related safety issues relative to conventional regimens. Furthermore, belatacept typically requires infusion for its administration chronically, which may present an inconvenience to patients.

Immunosuppressive effect of ASP2408, a novel CD86-selective variant of CTLA4-Ig, in rats and cynomolgus monkeys.

The CTLA4-Ig fusion proteins abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) costimulatory ligands and are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplantation, respectively. Abatacept and belatacept preferentially bind CD80, yet CD86 has been implicated as the dominant ligand for CD28-mediated costimulation of T cells. We investigated the immunosuppressive effects of ASP2408, a novel CTLA4-Ig with CD86 selectivity and high potency created by directed evolution methods.

ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution.

The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles.

A pilot trial targeting the ICOS-ICOS-L pathway in nonhuman primate kidney transplantation.

Costimulation blockade with the B7-CD28 pathway-specific agent belatacept is now used in clinical kidney transplantation, but its efficacy remains imperfect. Numerous alternate costimulatory pathways have been proposed as targets to synergize with belatacept, one of which being the inducible costimulator (ICOS)-ICOS ligand (ICOS-L) pathway. Combined ICOS-ICOS-L and CD28-B7 blockade has been shown to prevent rejection in mice, but has not been studied in primates.

Polyamine depletion therapy in prostate cancer.

The prostate gland has among the highest level of polyamines in the body and prostate carcinomas have even greater elevated polyamine levels. These ubiquitous molecules synthesized by prostate epithelium are involved in many biochemical processes including cellular proliferation, cell cycle regulation, and protein synthesis. These properties have made polyamines a potential target for therapeutic intervention in diseases of excessive cell proliferation such as cancer.

Induction of apoptosis by aryl-substituted diamines: role of aromatic group substituents and distance between nitrogens.

A series of aromatic substituted diamines was synthesized and characterized for their cytotoxic profiles against human breast and prostate tumor cell lines. Following a structure function analysis of the effects of changes of the benzyl substituents and the distance between amino groups the most potent analogues were analyzed biologically and were shown to induce apoptosis. These compounds do not induce the enzyme SSAT or deplete intracellular polyamine levels, mechanisms demonstrated by other cytotoxic polyamine analogues.

Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO.

Polyamines are ubiquitous molecules with multiple intracellular functions. Cells tightly regulate their levels through feedback mechanisms affecting synthesis, intracellular conversion, and transport. Because polyamines have an important role in regulating cell growth, they are a target for cancer therapeutic development.

Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid.

Structure-activity relationships in the region of the phthalide ring of the inosine monophosphate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the lactone ring with other cyclic moieties resulted in loss of potency, especially for larger groups. Replacement of the ring by acyclic substituents also indicated a strong sensitivity to steric bulk.

A 69-kDa membrane protein associated with lipopolysaccharide (LPS)-induced signal transduction in the human monocytic cell line THP-1.

Lipopolysaccharide (LPS) triggers a wide range of cellular responses in mammalian cells. Several proteins, including CD14, have been reported to possess LPS binding capacity. However, the signal transduction molecule(s) and pathway(s) through which LPS induces cellular responses have not been identified.

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V alpha 1 gene.

Previous studies of the TCR alpha chain gene have located promoter elements 5' to the start of the various V alpha genes. The only fully characterized enhancer for the entire alpha chain gene (V, J and C genes) has been located approximately 3 kb from the 3' end of C alpha. We now report the existence of additional regulatory elements located in the introns of several murine V alpha genes (V alpha 1, V alpha 3 and V alpha B6.

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.

Phenotypic identification of specific and nonspecific suppressor T-cell populations involved in the in vivo response to alloantigen.

The monoclonal antibody 984.D4.6 (ma984) has been previously shown to recognize antigen-specific suppressor T-cells in an in vitro alloantigenic mixed-lymphocyte response system.

The immunosuppressant leflunomide inhibits lymphocyte progression through cell cycle by a novel mechanism.

Leflunomide is a novel and effective immunosuppressant that holds promise as a therapeutic agent, but the mechanism of action is unknown. Here we provide evidence that leflunomide is a general cytostatic agent for a wide range of cells. The IC50 for proliferation in transformed cell lines ranged from 2 to 16 microM.

lnterleukin-2 Stimulates the Development of Anergy via the Activation of Nonspecific Suppressor T Cells.

We have previously demonstrated that incubation of splenic lymphocytes from an unimmunized mouse with IL-2 IFN-α or γ resulted in the development of a population of nonspecific regulatory cells (Ts). These cells were shown to block the ability of lymphocytes to generate mixed-lymphocyte responses in vitro. In the studies reported here, we have investigated the cell populations involved in this phenomenon.

Transduction of the IFN-gamma signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity.

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7.

Involvement of two distinct regulatory T cell populations in the antigen-specific suppression of cytolytic T cell generation.

Alloantigen-specific, radiation-resistant T cells generated in mixed-lymphocyte cultures inhibited the generation of allospecific CTL responses in vitro. This regulatory T cell population was studied using mAb generated to Ag-specific suppressor factors that regulate the response to the synthetic terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Both monoclonal 984 D4.

Cytokine effects on the down regulation of cytolytic T cell responses in vitro.

Lymphocytes from mouse and men can be stimulated by a variety of cellular signals including concanavalin A and the interferons to generate cells capable of non-specific down regulation of the generation of immune responses. The studies reported here extend this finding to show that such regulatory cells are also generated during the course of a mixed lymphocyte response and following stimulation of the CD3 component of the T cell receptor with anti-CD3 antibody. Regulatory activity was assessed by the addition of putative regulatory cells to mixed lymphocyte cultures and measuring the generation of cytolytic T cell activity in these cultures.

Putative N-terminal sequence of murine soluble immune response suppressor (SIRS): significant homology with short neurotoxin 1.

Soluble immune response suppressor (SIRS) is a low-molecular-weight protein (approximately 10,000 daltons) produced by mitogen- or interferon-activated T lymphocytes that can block development of humoral or cell-mediated immune responses in vivo or in vitro. As previously reported, murine SIRS is heterogeneous, eluting in two broad peaks on high performance reverse phase chromatography as well as displaying several broad isoelectric point forms. A putative N-terminal 21 amino acid sequence has been obtained for one of the less hydrophobic isoforms, SIRS-alpha 7.

Antipeptide antibody specific for the N-terminal of soluble immune response suppressor neutralizes concanavalin A and IFN-induced suppressor cell activity in an in vitro cytotoxic T lymphocyte response.

Soluble immune response suppressor (SIRS) is a nonspecific immunosuppressive lymphokine produced by Lyt-2+ lymphocytes after exposure to Con A, IFN-alpha, or IFN-gamma. The N-terminal 21 residues of SIRS have recently been elucidated and antisera specific for this sequence have been raised in rabbits by using a synthetic peptide coupled to an immunogenic carrier protein. In a series of experiments, we have established that this antiserum blocks the suppressive activity of Con A- or IFN-activated suppressor cells.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate: generation of suppressive macrophages from treated animals.

At concentrations normally found in the spleen, macrophages from animals treated with O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 hr were previously shown to be immunosuppressive (Rodgers et al., 1985b). In addition, it was shown that macrophages from OOS-TMP-treated animals had a diminished capacity to present antigen (Rodgers et al.

Organophosphorus pesticide immunotoxicity: effects of O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems.

The time course of immunosuppression induced by acute treatment with O,O,S-trimethyl phosphorothioate (OOS-TMP), an impurity in technical formulations of malathion, was examined in female C57B1/6 mice. Both cell-mediated and humoral immune responses were examined and included allospecific cytotoxic T cells, proliferative response to mitogens, interleukin-2 production and antibody production to sheep red blood cells. OOS-TMP pretreatment led to a reversible suppression of the generation of cytotoxic T lymphocytes and antibody-secreting cells to sheep erythrocytes.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate. II. Effect on the ability of murine macrophages to present antigen.

Acute administration of 10 mg/kg O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 h has been shown to suppress the in vitro generation of cytotoxic T lymphocyte responses and antibody-secreting cells to sheep red blood cells and to increase interleukin-2 production. Macrophages were shown to be the splenic cell population most affected by OOS-TMP pretreatment. In this report, the ability of macrophages from OOS-TMP-treated animals to function in antigen presentation was shown to be significantly decreased.