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Biomedical subjects

R J Robb

Publications and source records attributed to R J Robb.

At least 19 recordsLinked to original sources
PubMed2001

In situ vaccination against a non-immunogenic tumour using intratumoural injections of liposomal interleukin 2.

Cancers appear to escape surveillance by the immune system at least in part because they fail to induce a protective immune response. Therapeutic vaccines based on specific tumour antigens and tumour cells modified ex vivo by genetic techniques are but two strategies being used to circumvent this problem. In this report, we describe a simple, yet effective alternative in which tumour-specific responses are induced by in situ administration of a well-characterized liposomal formulation of the cytokine interleukin 2 (IL-2). Using the non-immunogenic B16 melanoma model, intratumoural injections of liposomal IL-2 L(IL2), were shown to induce a long-lived immune response specific for the injected tumour. In conjunction with subsequent removal of the primary tumours by surgery, the injections increased mean survival to 57 days from a control value of 32 days and partially protected surviving mice against re-challenge with B16. L(IL2) induced an early infiltration of inflammatory cells within the tumours which was followed several days later by an influx of CD3+ T cells. The cellular influx and a coincident decrease in tumour growth were noted in both injected tumours and a second non-injected tumour on the same animal, thereby demonstrating the systemic nature of the immune response. Intratumoural injections of soluble IL-2 at the same dose failed to induce B16-specific cellular immunity or to prolong survival of the mice. Thus, liposomal formulation of the cytokine was fundamental to successful induction of immunity by this in situ vaccination regimen.

Animals↗
PubMed2001

The effect of cholesterol in a liposomal Muc1 vaccine.

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Interleukin-2-induced small unilamellar vesicle coalescence.

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (<or=25 nm). Intermediates (partially coalesced vesicles) within the forming multilamellar structures were identified by freeze-fracture electron microscopy and their presence was corroborated by differential scanning calorimetry. Several distinct steps were identified in the coalescence process. In the initial step, rhIL-2 rapidly bound to the DMPC SUVs. This was followed by a pH-dependent conformational change in the protein, as evidenced by an increase in tryptophan fluorescence intensity. The SUVs then aggregated in large clusters that eventually annealed to form closed MLVs. In this process over 90% of the rhIL-2 was bound to and incorporated within the multilamellar structures.

Calorimetry, Differential Scanning↗
PubMed2000

Biopharmaceutics of liposomal interleukin 2, oncolipin.

Oncolipin is a multilamellar liposomal (dimyristoyl phosphatidylcholine) formulation of interleukin 2 (IL-2) and human serum albumin (HSA) with distinct surface characteristics which may influence its biological activities. IL-2 and HSA were detected on the surface of the liposomes using specific antibody staining. Surface expression of IL-2 was also demonstrated by the observation that Oncolipin bound to cells expressing IL-2 receptors (IL-2R) containing alphabetagamma or betagamma subunits. Binding and internalization of Oncolipin by cells expressing alphabetagamma or betagamma receptor subunits was blocked by excess free IL-2 or a neutralizing antibody against the beta chain. The display of surface IL-2 on Oncolipin's liposomes was maintained in vivo after intravenous injection into mice. IL-2 was also present between the lipid bilayers of the multilamellar liposomes based on the unique physical characteristics detected by freeze fracture electron microscopy. The bulk of the liposome-associated IL-2 was released from the liposomes upon incubation at 37 degrees C in medium containing serum, indicating that the IL-2 was not irreversibly entrapped on or in the liposome structure. Thus, Oncolipin is receptor-targeted to activated T and NK cells by virtue of its surface expression of IL-2 and has the potential to release IL-2 following deposition within lymphoid organs. These properties may confer distinct advantages over soluble IL-2 for immunotherapy of cancer and viral diseases.

Animals↗
PubMed2000

A comparison of biodistribution of liposomal and soluble IL-2 by a new method based on time-resolved fluorometry of europium.

A novel method was developed to determine the pharmacokinetics and biodistribution of cytokines and lymphokines based on time-resolved fluorometry (TRF) of europium (Eu). The comparison of two formulations of IL-2 was used to illustrate the sensitivity and applicability of this method as well as to extend the information on the pharmacokinetics of liposomal IL-2 and soluble IL-2. The blood kinetics and biodistribution of liposomal and soluble IL-2 in lymphoid organs and kidneys as measured by TRF were similar to those determined by the radioisotopic method. In both instances, the formulation of IL-2 into liposomes increased its serum half-life and accumulation in reticuloendothelial and lymphoid organs. The increased sensitivity of the Eu/TRF method permitted the extension of observational time points and the analysis of biodistribution in organs such as lymph nodes and bone marrow. These results suggest that Eu-labelled proteins in conjunction with TRF offer a suitable alternative to radiolabelled proteins for pharmacokinetics and tissue distribution studies in animals. This method offers distinct advantages over traditional techniques employing radioistopes since it has greater sensitivity, no half-life limitations and no radioactive or hazardous waste disposal.

Animals↗
PubMed1998

Liposomal formulation of a self lymphoma antigen induces potent protective antitumor immunity.

We developed a liposome carrier for a model nonimmunogenic, self Ag. This carrier reproducibly converted lymphoma Ig into a potent tumor rejection Ag in mice. A single immunization induced protection against challenges representing 20 to 100 times the minimum lethal dose of parental tumor. This protective effect required minimal amounts of incorporated Ag and IL-2 and elicited specific Abs (compared with free Ag or liposomal control Ig which did not elicit any specific Abs); depletion experiments demonstrated a requirement for effector CD4+ and CD8+ T cells. Head-to-head comparisons, indicating superior potency and induction of specific T cell activation, distinguished liposomal from prototype, carrier-conjugated Ag. These results provide a strategy for formulating weak tumor or other clinically important Ags into vaccines.

Animals↗
PubMed1993

Identification of a direct interaction between interleukin 2 and the p64 interleukin 2 receptor gamma chain.

The interleukin 2 receptor (IL-2R) consists of at least two subunits, alpha and beta, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R gamma chain, and have suggested that gamma chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the gamma chain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipitated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R gamma-chain complex. These studies thus establish that the IL-2R gamma chain directly contributes to the IL-2-binding site, consistent with the hypothesis that gamma chain influences IL-2R affinity through its direct interaction with IL-2.

Amino Acid Sequence↗
PubMed1992

Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R.

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.

Animals↗
PubMed1991

Internalization and nuclear localization of interleukin 1 are not sufficient for function.

The human fibroblast interleukin 1 (IL-1) receptor is a glycosylated transmembrane protein with a cytoplasmic domain of 213 amino acids. We have constructed a series of deletion mutants of the cytoplasmic region of the IL 1 receptor and have used these mutants to examine its role in ligand binding, internalization, signal transduction, and nuclear localization of IL-1. Mutant receptors lacking most of the cytoplasmic domain are expressed at the cell surface and can bind, internalize, and localize IL-1 at the nucleus, but they do not allow IL-1-mediated induction of interleukin 2 and SV40 promoters. We have localized a critical region for signal transduction to a 50-amino acid segment of the cytoplasmic domain of the receptor. These studies demonstrate that IL-1 internalization and nuclear localization are not sufficient to trigger IL-1 activation of gene expression in T-cells.

Autoradiography↗
PubMed1991

Quantitative characterization of the intrinsic ligand-binding affinity of the interleukin 2 receptor beta chain and its modulation by the alpha chain and a second affinity-modulating element.

The interleukin 2 receptor is a multisubunit receptor known to consist of at least two IL-2 binding subunits, alpha and beta. We report here kinetic evidence defining the contribution of an affinity-modulating element(s) intimately involved in modulation of the ligand-binding affinity of the beta chain and alpha/beta complex. The principal effect of this modulating element on the beta chain is to slow the dissociation of IL-2 more than 150-fold and thus raise its low intrinsic IL-2 binding affinity (Kd = 70 nM) as defined in transfected fibroblast cells to the level observed in lymphoid cells (Kd = 1.2 nM). The alpha subunit also increases the ligand-binding affinity of the beta chain, although in this case principally by increasing the association rate constant more than 1200-fold. The additional effect of the affinity-modulating element on the alpha/beta complex is minimal with regards to the equilibrium binding affinity. It does, however, have a detectable 14-fold effect on slowing the IL-2 dissociation rate. The existence of multiple forms of IL-2 receptor complexes with widely varying ligand affinities and dissociation rates illustrates the need for careful evaluation of binding data in studies of receptor subunit composition and reconstitution.

Animals↗
PubMed1990

Increased expression of the interleukin 2 (IL-2) receptor beta chain (p70) on CD56+ natural killer cells after in vivo IL-2 therapy: p70 expression does not alone predict the level of intermediate affinity IL-2 binding.

The expression of the 70-kD beta subunit of the interleukin 2 receptor (IL-2R) has been examined on peripheral blood lymphocytes (PBL) obtained from patients receiving systemic infusions of IL-2. Using monoclonal antibodies directed against p70, flow cytometric analyses revealed a greater than threefold increase in expression of the IL-2R beta chain on CD56+ natural killer (NK) cells from post-IL-2 therapy PBL relative to pre-therapy cells. The level of p70 expression on the post-therapy cells was three- to fourfold greater (based on fluorescence intensity) than the level of p70 expression on YT cells, an NK-like cell line that expresses approximately 12,000 intermediate affinity IL-2 binding sites/cell. Despite the high level of p70 expression, in 125I-IL-2 binding assays only 790-1,290 intermediate affinity IL-2 binding sites/cell were detected on post-therapy cells from six patients. These data represent the first report of increased p70 expression after in vivo IL-2 administration and suggest a requirement for at least one additional subunit for the formation of functional intermediate affinity IL-2Rs. Furthermore, the presence on the surface of post-therapy NK cells of excess p70 that does not bind IL-2 with intermediate affinity implies that the formation of intermediate affinity IL-2Rs is not solely determined by the level of p70 expression, and that the response of NK cells to IL-2 might be regulated by altering the expression of p70 or some other IL-2R subunit.

Antibodies, Monoclonal↗
PubMed1990

Functional expression of native and chimeric human and murine IL-2 receptor p70 chains in an IL-3-dependent murine cell line.

Full length cDNAs encoding the human and murine p70 genes were isolated using polymerase chain reaction (PCR) and conventional cDNA library screening techniques, respectively. To validate their functional potential, expression vectors containing human, murine and chimeric human/murine p70 cDNAs were transfected into the murine IL-3-dependent cell line FDC-P1. Transfected cells expressed a combination of high and low-affinity IL-2 binding sites while parental cells displayed only the low-affinity sites associated with expression of endogenous p55 IL-2 receptor chains. The role of the transfected p70 chains in formation of the high-affinity receptor sites was confirmed by the finding that the species-specific inhibitory antibody TU27 blocked high-affinity binding to human p70 and chimeric human/murine p70-expressing cells while having no effect on cells transfected with the murine p70 cDNA construct. As consequences of the expression of the transfected p70 chains, the cells responded to IL-2 with increased levels of endogenous p55 receptor subunit and both short and long-term proliferation. These studies substantiate the role of the p70 receptor chain in functional responses to IL-2 and provide a model system for future dissection of the structure/function relationships of the IL-2 receptor.

Animals↗
PubMed1988

The construction and characterization of a biologically active recombinant IL-2 containing a lysine-rich C-terminal extension.

A polylysine extended gibbon IL-2 (IL-2-L) was constructed by the addition of a lysine-rich oligopeptide, Gly3-(Lys-Lys-Asp)3-Leu-Glu to the C terminus of gibbon IL-2 by using rDNA technology. The bioactivity of the hybrid molecule was comparable to that of normal human rIL-2 in assays measuring T cell proliferation, and in generation of lymphokine-activated killer cells from PBL. Furthermore, the addition of the lysine-rich segment made the molecule more amenable to the chemical derivatization of amino groups. After biotinylation, IL-2-L retained a greater proportion of its bioactivity than normal rIL-2. In addition, biotinylated IL-2-L was capable of simultaneously binding to cell surface IL-2R and streptavidin. Thus, the addition of the lysine-rich oligopeptide facilitated the generation of an active form of biotinylated IL-2 which acts as a bridge between IL-2R-positive cells and avidin-coupled reagents and affinity supports. Such a selective means of labeling and binding IL-2-responsive cells may have great practical utility for IL-2-based immunotherapy.

Amino Acid Sequence↗
PubMed1988

Structure-function relationships for the IL-2 receptor system. V. Structure-activity analysis of modified and truncated forms of the Tac receptor protein: site-specific mutagenesis of cysteine residues.

IL-2R on activated lymphocytes contain the Tac protein. As part of an effort to characterize this molecule, we examined the structure-activity relationship for each of its 12 Cys residues. A preliminary map of intramolecular disulfide bonding was derived by analysis of cystine-linked enzymatic fragments of the Tac protein. The results indicated that disulfide bonds linked Cys-3 with Cys-147, Cys-131 with Cys-163, and Cys-28,30 with Cys-59,61. The contribution of the Cys residues to an active protein conformation was tested by site-specific mutagenesis, followed by expression of the modified molecules in murine L cells. The results indicated that Cys-192 and -225 could be replaced without affecting ligand binding. In contrast, modification of any of the other 10 Cys residues, either singly or in combinations corresponding to the predicted disulfide bonds, greatly reduced the ability of the corresponding protein to bind IL-2 or either of two mAb (anti-Tac and 7G7/B6) which recognize the Tac protein. Each of the latter mutations also interfered with the molecule's post-translational modification and cell-surface expression. Consistent with these findings, transfection of the L cells with vectors containing truncated Tac cDNA inserts resulted in secretion of Tac fragments capable of ligand binding when the polypeptide chains terminated after Cys-163 (the 10th Cys residue in the full length molecule), but resulted in inactive fragments of Tac which were poorly secreted when they terminated before Cys-163. These findings emphasize the remarkable sensitivity of the active conformation of the Tac molecule to each of the postulated intramolecular disulfide bonds.

Amino Acid Sequence↗
PubMed1988

Structure-function relationships for the interleukin 2 receptor: location of ligand and antibody binding sites on the Tac receptor chain by mutational analysis.

The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of three monoclonal antibodies (anti-Tac, GL439, and H31) that block high- and low-affinity Tac-IL-2 interactions, thus supporting the hypothesis that these segments of the protein are at or near sites of receptor-ligand contact. In contrast, the apparent binding sites of antibodies (Hiei and H47) that selectively inhibit high-affinity IL-2 binding were mapped to a distinct location (residues 158-160) within the region encoded by exon 4 of the Tac gene. Since high-affinity receptors consist of a heterodimer of Tac and a second ligand-binding protein (p70), this portion of the Tac molecule may be involved in the interaction between the two receptor subunits. As expected, the binding sites of noninhibitory antibodies (7G7/B6, residues 140-144; H48, residues 170-211) did not overlap those segments in which IL-2-binding mutants were observed. These results provide a preliminary correlation of structure and function for the Tac protein that should prove useful in evaluating detailed models of the IL-2-receptor complex.

Animals↗
PubMed1987

Structure-function relationships for the IL 2-receptor system. IV. Analysis of the sequence and ligand-binding properties of soluble Tac protein.

The Tac protein is one of at least two glycoproteins known to bind the growth and differentiation factor interleukin 2 (IL 2). In addition to its location on the cell surface, where it plays a part in high and low affinity IL 2 receptors, Tac is released from activated lymphocytes in a soluble form. We observed this release both for Tac protein labeled biosynthetically and for Tac protein labeled by surface iodination of intact cells. Competitive binding studies indicated that the soluble Tac protein retained an ability to bind IL 2 with a low affinity (Kd of 11.1 nM). In addition, structural analysis revealed that the polypeptide chain began at position 1 and ended at or just before Cys-192 of the full-length molecule. Thus, the protein was missing its normal transmembrane and intracytoplasmic segments, accounting for its solubility and cellular release. The apparent lack of modification in the amino acid sequence and the termination at Cys-192 are inconsistent with a mechanism of cellular release dependent only on alternate mRNA splicing. Instead, the results suggest that proteolysis may accompany the release of soluble Tac protein from cells expressing IL 2 receptors.

Amino Acid Sequence↗
PubMed1987

Structure function relationships for the IL 2 receptor system. III. Tac protein missing amino acids 102 to 173 (exon 4) is unable to bind IL 2: detection of spliced protein after L cell transfection.

High affinity receptors for interleukin 2 (IL 2) contain the Tac protein as one ligand-binding subunit. Localization of the IL 2-binding site on this molecule, as well as localization of the complementary site on IL 2, should provide insight into the design of IL 2 analogs. In this report, we examine the ability of normal and modified Tac protein to bind IL 2 and several antibodies that recognize the native Tac molecule. Using a transient L cell expression system, we have determined that transfection with cDNA-missing Tac exon 4 resulted in expression of spliced protein that had no measurable binding to IL 2 or the monoclonal anti-receptor antibodies, anti-Tac, and 7G7/B6. This protein was detected, however, by rabbit polyclonal antibodies prepared against synthetic Tac peptides. Thus, one or more amino acids encoded by exon 4 is important, either for direct ligand contact or for the proper folding of critical segments of the Tac molecule. In addition, insertion of stop codons at a unique restriction enzyme site near the beginning of exon 5 resulted in cellular secretion of truncated Tac molecules that were capable of binding IL 2, anti-Tac, and 7G7/B6. Amino acids encoded by exons 5 to 8 thus play no critical role in IL 2 binding. The ligand association demonstrated for truncated Tac protein produced by exons 2 to 4 should guide attempts to define the IL 2-binding segment of the Tac molecule.

Amino Acid Sequence↗