Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo.

Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist.

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al.

Humanized antibody directed to the IL-2 receptor beta-chain prolongs primate cardiac allograft survival.

IL-2Rs are expressed by T cells activated in response to foreign histocompatibility Ags but not by normal cells. This difference in IL-2R expression is exploited by blockade of IL-2Rs to achieve immunosuppression. High affinity IL-2Rs involve three subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma.

Consensus repeat domains of E-selectin enhance ligand binding.

To study the structural characteristics of E-selectin necessary for mediating cell adhesion, we examined the role of the consensus repeat (CR) domains in E-selectin function. Soluble constructs containing different numbers of CR domains were stably expressed in Chinese hamster ovary cells, purified to homogeneity, and characterized. The minimum functional unit of soluble E-selectin consisted of the lectin (Lec) and epidermal growth factor (EGF) domains alone (Lec-EGF) as indicated by its ability to mediate in vitro HL-60 cell adhesion.

Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains.

The three-dimensional structure of the ligand-binding region of human E-selectin has been determined at 2.0 A resolution. The structure reveals limited contact between the two domains and a coordination of Ca2+ not predicted from other C-type lectins.

Endothelial monocyte-activating polypeptide II. A novel tumor-derived polypeptide that activates host-response mechanisms.

An important means by which tumor cells influence the vasculature is through the production of soluble mediators altering vascular properties. A approximately 22-kDa polypeptide was purified to homogeneity from conditioned medium of murine methylcholanthrene A (meth A) fibrosarcoma cells by ion-exchange chromatography and preparative sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), based on its ability to induce tissue factor procoagulant activity in endothelial cells (ECs). The final product migrated as a broad band on reduced and nonreduced SDS-PAGE and had an unique amino-terminal sequence.

Cloning and expression of murine IL-12.

Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively).

Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2).

Reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac in cynomolgus monkeys.

The anti-Tac mAb has been shown to bind to the p55 chain of the IL-2R, block IL-2 binding and inhibit T cell proliferation. A humanized form of anti-Tac (HAT) has been constructed that retains the binding properties of murine anti-Tac (MAT). These two mAb were evaluated in cynomolgus monkeys to compare relative immunogenicity and pharmacokinetic properties.

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation.

Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.

Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells.

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration.

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF.

Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human B-lymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.

Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins.

Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration.

A polypeptide factor produced by fibrosarcoma cells that induces endothelial tissue factor and enhances the procoagulant response to tumor necrosis factor/cachectin.

Intravascular clot formation, localized to the neoplasm, is an early component of the vascular response to tumor necrosis factor (TNF)/cachectin. Fibrin is closely associated with the endothelial cell surface, and multiple microthromboses lead to reduced blood flow in the tumor. We have identified a tumor-derived mediator which enhances endothelial procoagulant activity and the cellular response to TNF using cultured cells derived from a murine methylcholanthrene A (meth A)-induced fibrosarcoma as a model system.

Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor.

Recombinant technology has facilitated the production of two soluble forms of human interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two IL-2Rs, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration.

Characterization of a factor(s) which synergizes with recombinant interleukin 2 in promoting allogeneic human cytolytic T-lymphocyte responses in vitro.

Supernatants from PHA-activated human peripheral blood mononuclear cells, depleted of virtually all IL-2 activity by an anti-rIL-2 immunoadsorbent column, contain a factor(s) which synergizes with rIL-2 in facilitating the generation of allogeneic human CTL responses in vitro. This factor, provisionally termed CTL maturation factor (TcMF), did not appear to promote CTL responses in the absence of rIL-2. Furthermore, it acted later than IL-2 in facilitating CTL responses and could not be replaced by recombinant IFN-gamma.

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction.

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification.

Inhibition of spontaneous proliferation of human leukemic B cells from patients with chronic lymphocytic leukemia by anti-mu antibodies.

In a monoclonal system, F(ab')2 fragments of rabbit anti-mu antibody (anti-mu) were found to inhibit the proliferation and differentiation of highly purified E-rosette-negative largely leukemic B cells from two patients with chronic lymphocytic leukemia (CLL). Leukemic B cells from these two patients, in contrast with the majority of patients with CLL, exhibited high spontaneous proliferation in culture medium alone. This spontaneous proliferation was significantly inhibited by moderate concentrations of anti-mu (10-50 micrograms/ml).

Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase.

Placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.

Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus.

Natural human interferon gamma(IFN-gamma) was purified from the conditioned medium of peripheral blood leukocytes using selective silica gel adsorption and antibody-affinity chromatography. SDS-PAGE and Western blot analysis demonstrated three major species with molecular masses of 25 kDa, 20 kDa and 17 kDa. Structural analysis of this natural IFN-gamma preparation demonstrated a pyroglutamate residue at the amino terminus and a heterogeneous carboxyl terminus.

Recombinant human interleukin 1 alpha: purification and biological characterization.

Interleukin 1 (IL 1) is a polypeptide hormone produced by activated macrophages that affects many different cell types involved in immune and inflammatory responses. The cloning and expression of a murine IL 1 cDNA in Escherichia coli encoding a polypeptide precursor of 270 amino acids has been reported, and expression of the carboxy-terminal 156 amino acids of this precursor in E. coli yields biologically active IL 1.

Isolation of proteins from crude mixtures with silica and silica-based adsorbents.

Silica and silica-based adsorbents have been used to isolate specific proteins from crude mixtures. Acid-activated Nugel silica and Nugel SP-Silica were used to extract human immune interferon from the conditioned medium of mixed lymphocyte cultures. Human interleukin-2 was extracted from the conditioned medium of Jurkat cells with LiChroprep RP-8.

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons.

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer.