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R P Junghans

Publications and source records attributed to R P Junghans.

At least 19 recordsLinked to original sources
PubMed1999

Bypassing immunization: optimized design of "designer T cells" against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA.

Tumor-associated antigens are typically nonimmunogenic in cancer patients, "immune surveillance" having manifestly failed. The fact that most tumor antigens are normal human proteins presents significant obstacles to current cancer immunization approaches that researchers are presently striving to overcome. An alternative strategy bypasses immunization altogether by direct genetic alteration of autologous patient T cells, to create "designer T cells" specific to a particular antigen. Chimeric immunoglobulin-T cell receptors (IgTCR) with a specificity for carcinoembryonic antigen (CEA) were created to evaluate the optimal IgTCR structure for cancer therapy. Antigen-binding domains of a humanized antibody were combined with TCR signaling chains to yield four different chimeric IgTCR: single chain Fv fragment (sFv)-zeta, fragment antigen-binding (Fab)-zeta, sFv-epsilon, and Fab-epsilon. All of the IgTCR were well expressed on T cells, and all showed specific binding and activation, as demonstrated by IL-2 production on contact with immobilized or cellular CEA, excepting sFv-epsilon alone which was inert solely against cellular targets for steric reasons unique to this construct. In contrast to prior studies of isolated TCR chains that related increased tyrosine-based activation motifs in zeta as a reason for superior signaling potency, these tests are the first to show that epsilon and zeta are indistinguishable for T cell signaling when assayed in the context of the intact TCR complex. Further, Fab was equivalent to sFv as an IgTCR component for expression and antigen binding, establishing an important alternative for IgTCR antigen recognition because sFvs may often lose antigen affinity. When IgTCR was expressed on normal human T cells, cytotoxic potency was demonstrated at low E:T ratios, with T cell recycling and progressive tumor cell destruction. Contrary to recent speculations, these observations prove that high affinity TCR interactions are not an impediment to serial target engagement and disengagement by cytotoxic T cells. The multivalent intercellular interactions of target cell binding, activation, and cytotoxicity were resistant to inhibition by soluble CEA. These studies establish a potentially important new immunotherapeutic modality for the treatment of CEA-expressing tumors.

CD3 Complex↗
PubMed1998

Measurement and biological correlates of antibody bioactivity during antibody immunotherapies.

An important factor in the effectiveness of antibody immunotherapies is the antibody bioactivity, or the availability of free binding sites. Bioactivity may be decreased in settings where the target antigen exists in a soluble form capable of binding to circulating antibody. Because many antigens have soluble forms, we developed a method for determining if antibody is bound by soluble antigen in vivo. As a model system, we studied the interaction of soluble interleukin-2 receptor alpha (Tac; IL2R alpha; CD25) and anti-Tac antibody. We show first that HPLC readily separates free antibody from antibody which is monovalently or bivalently bound by soluble antigen. Further, we demonstrate that the distribution of the three forms of antibody accords with predictions of mass action and the binomial probability distribution. These methods were used to examine the bioactivity and concentration of free antibody in 14 patients undergoing therapeutic trial with Humanized anti-Tac antibody in leukemia and lymphoma. Results of two contrasting patients are highlighted. Low bioactivities correlated with reduced targeting of tumor cells and reduced therapeutic effectiveness. This report highlights the importance of soluble antigen in antibody therapies and demonstrates a simple method for evaluating in vivo bioactivity of antibody after therapeutic administration.

Adult↗
PubMed1998

Impact of antigenemia on the bioactivity of infused anti-Tac antibody: implications for dose selection in antibody immunotherapies.

In patients with malignancies and immune disorders expressing Tac (alpha chain of the interleukin 2 receptor; CD25), physiologic shedding of this receptor may lead to high blood levels of the soluble form (sTac). This system was used to model the interaction of soluble antigen with antibody in therapeutic settings and to develop rational principles to optimize the delivery of antibody to tumor target cells. First, we confirmed that sTac in vivo can block anti-Tac binding sites and diminish antibody binding to Tac+ cells. Second, the bioactivity of antibody in vivo correlated directly with the amount of antibody infused and inversely with the sTac concentration. Third, bindability of antibody declined in the hours and days after anti-Tac infusion in patients. Finally, tumor targeting was achieved even in the presence of excess sTac, demonstrating a partition of antibody between soluble and cell-bound antigen. A role is proposed for the Brambell receptor (FcRB) to delay saturation of human or chimeric antibodies via differential catabolism of antigen-antibody complexes. Principles are developed for predicting activity of administered antibody in the presence of soluble antigen to assist in dose selection in passive, radioimmuno and immunotoxin therapies.

Biological Availability↗
PubMed1998

Examination of a role for idiotypy in the disease remission of a long-term survivor of adult T cell leukemia treated with anti-Tac antibody.

The alpha chain of the interleukin 2 receptor (IL2R alpha; Tac) was targeted in clinical trials with adult T cell leukemia using murine anti-Tac antibody. Of 19 patients, a single individual achieved a durable complete remission. The mechanism of this action by murine anti-Tac has not been defined. We examined the hypothesis that the maintenance of the long-term response after treatment might be related to induction of a network of anti-idiotypic antibodies, as proposed in other tumor settings. In contrast to anti-Tac non-responders, the patient was found to have produced a human anti-mouse antibody (HAMA) response, and specifically an anti-idiotypic (Ab2) response, that was readily detectable by standard assays 4 years after treatment. Using phage display antibody libraries, this response was shown to be monoclonal, consisting of a single IgG1,kappa antibody of moderate affinity. No evidence was found for anti-anti-idiotypic (Ab3) antibodies with reactivity for sTac, which might alternatively have maintained an autogenic human anti-Tac antibody response. An area of limited homology was noted between the Ab2 antibody and the IL2R in the domain of IL2 binding, but no binding of Ab2 to IL2 could be shown that might have reduced endogenous ligand (IL2) concentrations. Similarly, no anti-anti-idiotypic (T3) T cell response was detected. Thus, we are unable to confirm features of idiotypy that could suggest a role in maintaining an anti-tumor response by anti-Tac antibody therapy.

Aged↗
PubMed1998

A single-chain immunotoxin against carcinoembryonic antigen that suppresses growth of colorectal carcinoma cells.

We have engineered an anti-carcinoembryonic antigen (CEA) single-chain immunotoxin derived from humanized anti-CEA antibody (hMN14) and a truncated Pseudomonas exotoxin (PE), PE40. The purified anti-CEA immunotoxin (hMN14(Fv)-PE40) was first measured for binding affinity against a CEA-positive colorectal carcinoma cell line and compared with its parental IgG and the monovalent Fab fragment. The Ka of sFv-PE40, Fab, and IgG were 5 x 10(7), 6 x 10(7), and 3 x 10(8) M(-1), respectively. There was no significant affinity loss by conversion of Fab to the single-chain Fv, but these monovalent forms were 5-6-fold reduced in affinity compared with the parental IgG. In cytotoxicity assays, the hMN14(Fv)-PE40 showed specific growth suppression of CEA-expressing colon cancer cell lines MIP-CEA (high CEA) and LS174T (moderate CEA) with IC50s of 12 ng/ml (0.2 nM) and 69 ng/ml (1.1 nM). These IC50s correlated inversely with the surface expression of CEA, such that 50% killing was equivalent for each cell type when expressed in toxin molecules bound/cell (3000-5000). The presence of soluble CEA up to 1000 ng/ml did not affect the cytotoxicity against CEA-expressing cells, with 50% suppression only at 4000 ng/ml that correlated with the binding Kd of the single-chain Fv. The stability of the hMN14(Fv)-PE40 molecule at 37 degrees C was confirmed by bioassay and by lack of aggregation. Our hMN14(Fv)-PE40 may be clinically useful for tumors with high CEA expression without affecting normal tissues with low or absent CEA, even in patients with high soluble antigen levels.

ADP Ribose Transferases↗
PubMed1997

Finally! The Brambell receptor (FcRB). Mediator of transmission of immunity and protection from catabolism for IgG.

F. W. Rogers Brambell was the father of the field of transmission of immunity, which he entered 50 years before the present era. As part of his quantitative and temporal studies on transmission, he defined the first Fc receptor system for IgG, and furthermore recognized the link between transmission of passive immunity from mother to young and protection from catabolism for IgG. This article provides a historical overview of the efforts of Professor Brambell and summarizes the subsequent elaboration of the details of the physiology and molecular biology of this remarkable receptor system.

Allergy and Immunology↗
PubMed1997

An optimized method for cell-based phage display panning.

BACKGROUND: Traditional methods of phage display panning, bind purified antigen to plates or other solid phases to which libraries are then applied, followed by vigorous washings in detergent-supplemented buffers to select for specific phage Fab. These methods are not directly applicable to antigens in their native environment on cell surfaces or in settings where the target antigen is unknown. OBJECTIVES: To develop a model antigen system employing whole cells rather than purified protein immobilized on a substrate; to optimize methods for phage display panning using a cell-based system. RESULTS: Specificity of binding of phage Fab to antigen on cells was demonstrated by output titer and by flow cytometry. Output titers showed a plateau and binding advantage, after four washes, corresponding to removal of most non-specifically bound phage. Enrichment advantage was independent of input phage number. Longer incubation times, to cell tolerance, improved specific binding. Temperature had modes impact as a variable in the panning and washing. An increase in output titers paralleled enrichment for specific phage Fab. CONCLUSION: An optimized method applied to whole cells can productively enrich specific phage Fab in mixtures with large excesses of non-specific phage Fab over several rounds of panning.

Animals↗
PubMed1996

Optimization of radioligand binding and radioimmuno assays.

Radioligand binding assay (RLA) and radioimmunoassay (RIA) development involves consideration of a large number variables which influence signal readout or system errors, with contrary effects on assay performance. We have developed an optimization strategy according to statistical criteria which incorporates these variables and which specifies a minimum data set for a systematic analysis. Increased background and error fractions are seen to reduce assay power, whereas increased specific activity, affinity, and counting time increase power. The optimal concentration of radiolabeled developer in the test is dependent upon a balance of these factors, but in general yields a broad optimum. These analyses suggest several strategies available to the investigator for improving assay performance. The optimization method is adapted to standard microcomputer spreadsheet formats which allow ready application of these procedures in any research laboratory.

Models, Chemical↗
PubMed1996

The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor.

More than 30 years ago, Brambell published the hypothesis bearing his name [Brambell, F. W. R., Hemmings, W. A. & Morris, 1. C. (1964) Nature (London) 203, 1352-1355] that remains as the cornerstone for thinking on IgG catabolism. To explain the long survival of IgG relative to other plasma proteins and its pattern of increased fractional catabolism with high concentrations of IgG, Brambell postulated specific IgG "protection receptors" (FcRp) that would bind IgG in pinocytic vacuoles and redirect its transport to the circulation; when the FcRp was saturated, the excess unbound IgG then would pass to unrestricted lysosomal catabolism. Brambell subsequently postulated the neonatal gut transport receptor (FcRn) and showed its similar saturable character. FcRn was recently cloned but FcRp has not been identified. Using a genetic knockout that disrupts the FcRn and intestinal IgG transport, we show that this lesion also disrupts the IgG protection receptor, supporting the identity of these two receptors. IgG catabolism was 10-fold faster and IgG levels were correspondingly lower in mutant than in wild-type mice, whereas IgA was the same between groups, demonstrating the specific effects on the IgG system. Disruption of the FcRp in the mutant mice was also shown to abrogate the classical pattern of decreased IgG survival with higher IgC concentration. Finally, studies in normal mice with monomeric antigen-antibody complexes showed differential catabolism in which antigen dissociates in the endosome and passes to the lysosome, whereas the associated antibody is returned to circulation; in mutant mice, differential catabolism was lost and the whole complex cleared at the same accelerated rate as albumin, showing the central role of the FcRp to the differential catabolism mechanism. Thus, the same receptor protein that mediates the function of the FcRn transiently in the neonate is shown to have its functionally dominant expression as the FcRp throughout life, resolving a longstanding mystery of the identity of the receptor for the protection of IgG. This result also identifies an important new member of the class of recycling surface receptors and enables the design of protein adaptations to exploit this mechanism to improve survivals of other therapeutic proteins in vivo.

Animals↗
PubMed1996

Biophysical characterization of a recombinant soluble interleukin 2 receptor (Tac). Evidence for a monomeric structure.

The interleukin 2 receptor (IL2R) plays a prominent role in the biology of T cells, B cells, and NK cells during activation. Of the three chains described, the alpha-chain of the receptor (Tac; IL2R alpha; CD25) is the most subject to regulation and is shed from the surface of activated cells to generate a soluble form in serum and tissues. Conflicting results have been reported on the native structure of soluble Tac, suggesting variously a monomer, a dimer, or higher noncovalent forms, spawning different models for its mechanism of action. We similarly show a large M(r)(app) by HPLC sieving chromatography, suggesting a tetrameric form. However, stoichiometry-ordered size (SOS) analysis of antibody-antigen complexes indicated only a single epitope per Tac molecule, compatible with a monomeric form. This larger M(r)(app) also conflicted with prior in vivo data showing rapid filtration of soluble Tac through the renal glomerulus that was not expected of a larger complex. Using different solvents, denaturants, and columns in the chromatography suggested that the elevated M(r)(app) values were an artifact of solute-column interactions, termed "ionic exclusion", rather than reflecting larger native structures. Analytical ultracentrifugation using a new type of analysis specific to glycoproteins demonstrated monomeric masses under all salt conditions with no tendency to form dimers or higher aggregates. Finally, circular dichroism spectroscopy showed no salt-dependent changes to suggest conformational alterations that might correlate with mobility changes on high pressure liquid chromatography. We conclude therefore that Tac is monomeric under physiologic conditions. Assessments of higher molecular weight for the purified soluble protein by other methods may be explained by the highly acidic nature of the molecule, which hampers matrix penetration with chromatographic media and by the high carbohydrate content and low partial specific volumes that accelerate the molecule in sedimentation media relative to pure protein standards.

Chromatography, Gel↗
PubMed1996

Metabolism of Tac (IL2Ralpha): physiology of cell surface shedding and renal catabolism, and suppression of catabolism by antibody binding.

The interleukin 2 receptor alpha (IL2Ralpha; CD25; Tac) is the prototypic model for soluble receptor studies. It exists in vivo as a transmembrane complete molecule (TM-Tac) on cell surfaces and as a truncated soluble form (sTac; sIL2R alpha). sTac has been used as a serum marker of T cell activation in immune disorders and of tumor burden in Tac-expressing malignancies. In vivo, serum levels of all soluble proteins depend on the balance between production and catabolism, but little is known about the metabolic features of this class of molecules. We have developed a model for Tac metabolism that incorporates new insights in its production and catabolism. Tac was shed from the surface of malignant and activated human T cells with a model half-life (t1/2) of 2-6h, but which was prolonged under certain circumstances. The rate of shedding is first order overall and nonsaturable over a two order of magnitude range of substrate (TM-Tac) expression. Once shed from cells Tac is subject to catabolic activities in the host. In vivo studies in mice showed that 90% of Tac was catabolized by the kidney with a t1/2 of 1 h and a filtration fraction of 0.11 relative to creatinine. The remaining 10% of catabolism was mediated by other tissues with a t1/2 of 10 h. Approximately 1-3% of sTac is excreted intact as proteinuria with the remaining 97-99% catabolized to amino acids. Antibody to the receptor induced a marked delay in sTac catabolism by preventing filtration of the smaller protein through the renal glomerulus and additionally suppressing other nonrenal catabolic mechanisms. A discrepancy between the catabolic rats for Tac and anti-Tac in the same complex was interpreted as a previously unrecognized differential catabolic mechanism, suggesting features of the Brambell hypothesis and immunoglobulin G transport and catabolism, in which the antigen-in-complex in intracellular vesicles is relatively less protected from catabolism than the associated antibody. In light of the pivotal role played by the kidney in sTac catabolism and the impact of administered antibody, the serum concentration of Tac in the settings of renal dysfunction or antibody therapy is not a suitable surrogate of activated T cells or of the body burden of tumor. These results provide parameters for assessing soluble receptor-ligand interactions generally.

Animals↗
PubMed1995

Radioimmunotherapy of interleukin-2R alpha-expressing adult T-cell leukemia with Yttrium-90-labeled anti-Tac.

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotropic virus-I. It is an aggressive leukemia with a median survival time of 9 months; no chemotherapy regimen appears successful in inducing long-term disease-free survival. The scientific basis of the present study is that ATL cells express high-affinity interleukin-2 receptors identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference, we administered anti-Tac armed with Yttrium-90 (90Y) to 18 patients with ATL initially (first 9 patients) in a phase I dose-escalation trial and subsequently (second group of 9 patients) in a phase II trial involving a uniform 10-mCi dose of 90Y-labeled anti-Tac. Patients undergoing a remission were permitted to receive up to eight additional doses. At the 5- to 15-mCi doses used, 9 of 16 evaluable patients responded to 90Y anti-Tac with a partial (7 patients) or complete (2 patients) remission. The responses observed represent improved efficacy in terms of length of remission when compared with previous results with unmodified anti-Tac. Clinically meaningful (> or = grade 3) toxicity was largely limited to the hematopoietic system. In conclusion, radioimmunotherapy with 90Y anti-Tac directed toward the IL-2R expressed on ATL cells may provide a useful approach for treatment of this aggressive malignancy.

Adult↗