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.

Sorting vector producer cells for high transgene expression increases retroviral titer.

Vector producer cells are derived from helper cell lines expressing viral proteins that have been transduced to express a transgene-carrying retroviral genome. Vector producing cells express two relevant forms of RNA in their cytoplasm: vector RNA (vRNA) that is packaged as the actual gene transfer agent, and messenger RNA (mRNA) from which transgene is translated. Two premises underlie this study: (1) vRNA is limiting for virus production and (2) mRNA is proportional to vRNA. Together, these premises predict that transgene expression in the vector producing cells will be predictive of the viral titer from those cells. In this case, sorting the vector producing cells for high transgene expression should select for more virus production in vector producing cell supernatants. This prediction was supported, with a greater than fivefold benefit in viral titer. This demonstrates a rapid and simple method by which to obtain significantly increased viral titers from the same vector producing cell preparation.

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.

Antileukemic effect of daclizumab in CD25 high-expressing leukemias and impact of tumor burden on antibody dosing.

Humanized anti-CD25 antibody, daclizumab, was applied in a pilot study of 10 patients with CD25(+) leukemias and pharmacokinetic/pharmacodynamic properties were characterized. Two widely held concepts - tumor sink accelerating pharmacokinetics and higher antigen expression correlating with target cell clearance - were supported by this first systematic evaluation of these questions with actual human clinical data. A flexi-dosing regimen was validated for maintaining target drug levels in vivo with a wide range of tumor burdens. Daclizumab induced clearance of peripheral leukemic cells when highly positive for CD25, but durable responses were not obtained. If daclizumab will have a role in antileukemic therapy, it may be in minimal disease settings or as a component of a combination regimen, but only when CD25 expression is high.

Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.

Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT-PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.

Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders.

The Mr 55,000 interleukin 2 receptor peptide (Tac; CD25) is not expressed by normal resting T-cells but is markedly up-regulated in adult T-cell leukemia and other malignancies, as well as on T-cells activated in normal immune, autoimmune, allograft, and graft-versus-host settings. Anti-Tac is a mouse monoclonal antibody directed against the Tac peptide. Our prior attempts to use this antibody in humans for antitumor therapy and immune regulation have been limited by weak recruitment of effector functions and neutralization by antibodies to mouse immunoglobulins. To circumvent these difficulties, we prepared several chimeric "humanized" anti-Tac antibodies by genetic engineering, including one "hyperchimeric" antibody (anti-Tac-II) in which the molecule is human except for the small hypervariable segments of the complementarity-determining regions retained from the mouse antibody. These constructs maintain high affinities for antigen and abilities to block T-cell activation and demonstrate new capabilities to perform antibody-dependent cell-mediated cytotoxicity, absent in the mouse anti-Tac. Hence, humanized antibodies have been developed to a tumor-associated antigen and activated T-cell marker with significant features that offer new therapeutic possibilities for select neoplastic and immune disorders.

Evidence for an antigen-driven humoral immune response in medullary ductal breast cancer.

A minority of breast cancers is characterized by lymphoplasmacytic infiltrates that have been correlated with improved patient survivals. The association of improved prognosis with plasmacytic infiltrates has been classically linked with the rare medullary carcinoma subtype but is also evident in the smaller infiltrated fraction of the more abundant nonmedullary (not otherwise specified) tumors. It is our hypothesis that these plasma cell (PC) infiltrates represent a host humoral response driven by one or more tumor-derived neoantigens. As the index study group, two primary medullary carcinoma tumors were examined. Immunophenotyping confirmed a large number of IgG PCs in contradistinction to normal breast, which typically contains a lesser number of mainly IgA isotypes. IgG heavy and light chains were expressed as combinatorial phage Fab libraries. VH and VL sequences showed a preponderance of clonal groups in both patients, as identified by germ-line gene usage and junctional mutation patterns. Panning of phage Fab libraries against purified antigens excluded Her2/neu and p53 as the eliciting antigen, and failure of clonal enrichment by cell panning suggested that the neoantigen was not membrane expressed or was expressed at low levels. Cognate, original VH+VL pairs were obtained by single cell PCR of tumor PCs, which showed overlap with the pooled IgG libraries. Tumor-derived IgG V genes exhibited mutational patterns that were consistent with antigenic selection and affinity maturation. Where examined, IgG1 was the predominant isotype, consistent with a T-dependent (i.e., protein) antigen. From these data, we infer that the breast tumor PC infiltrates of the medullary carcinoma subtype are compatible with an autogenic tumor neoantigen-driven humoral immune response.

Nature of the bifunctional chelating agent used for radioimmunotherapy with yttrium-90 monoclonal antibodies: critical factors in determining in vivo survival and organ toxicity.

One factor that is critical to the potential effectiveness of radioimmunotherapy is the design of radiometal-chelated antibodies that will be stable in vivo. Stability in vivo depends on the condition that both the chelate linkage and radiolabeling procedures not alter antibody specificity and biodistribution. In addition, synthesis and selection of the chelating agent is critical for each radiometal in order to prevent inappropriate release of the radiometal in vivo. In the present study, we compare the in vivo stability of seven radioimmunoconjugates that use different polyaminocarboxylate chelating agents to complex yttrium-88 to the mouse anti-human interleukin-2 receptor monoclonal antibody, anti-Tac. Chelate linkage and radiolabeling procedures did not alter the immunospecificity of anti-Tac. In order to assess whether yttrium was inappropriately released from the chelate-coupled antibody in vivo, iodine-131-labeled and yttrium-88 chelate-coupled antibodies were simultaneously administered to the same animals to correlate the decline in yttrium and radioiodinated antibody activity. The four stable yttrium-88 chelate-coupled antibodies studied displayed similar iodine-131 and yttrium-88 activity, indicating minimal elution of yttrium-88 from the complex. In contrast, the unstable yttrium-88 chelate-coupled antibodies had serum yttrium-88 activities that declined much more rapidly than their iodine-131 activities, suggesting loss of the radiolabel yttrium-88 from the chelate. Furthermore, high rates of yttrium-88 elution correlated with deposition in bone. Four chelating agents emerged as promising immunotherapeutic reagents: isothiocyanate benzyl DTPA and its derivatives 1B3M, MX, and 1M3B. All four isothiocyanate agents showed prolonged yttrium-88 vascular survival which was essentially identical to that of their iodine-131 activity with only minimum accumulation (1.4-1.8%/g) of the yttrium-88 injected dose into bone. Thus, these four chelating agents were very stable in vivo and suitable for yttrium-monoclonal antibody radioimmunotherapy.

Pharmacokinetics and bioactivity of 1,4,7,10-tetra-azacylododecane off',N'',N'''-tetraacetic acid (DOTA)-bismuth-conjugated anti-Tac antibody for alpha-emitter (212Bi) therapy.

A major factor that is critical to the potential effectiveness of alpha-emitter 212Bi radioimmunotherapy is the design of radiometal-chelated antibodies that will be stable in vivo. The chelate should bind the radiometal firmly to minimize release of the radionuclide from the monoclonal antibody-chelate complex. The present study examines a member of a new class of polyamine carboxylate chelating compounds, the DOTA ligands, for conjugating radiometal ions to antibody. Biocompatibility and stability are assessed with the anti-Tac monoclonal antibody that is directed against the human interleukin 2 receptors. The scientific basis for the clinical use of this antibody in radioimmunotherapy is that resting normal cells do not express the interleukin 2 receptor, whereas the receptor is expressed on the surface of certain neoplasms and by activated T-cells in select autoimmune diseases and in allograft rejection. First, we examined the impact of the labeling procedure and the presence of the chelate, DOTA, on antibody bioavailability and survival. Next, we studied the capacity of the antibody-chelate complex to retain radiobismuth. Coupling DOTA to antibody or adding Bi(III) to DOTA-coupled antibody did not disturb antibody immunoreactivity in in vitro binding studies. In addition, as analyzed by in vivo studies, DOTA-antibody dummy labeled with nonradioactive bismuth showed pharmacokinetics and tissue distribution identical to those of antibody not modified with DOTA. DOTA-anti-Tac charged with radioactive bismuth showed pharmacokinetics identical to radioiodinated dummy-labeled DOTA-antibody, suggesting little premature release of radioactive bismuth from the antibody complex. Moreover, in the early, therapeutically relevant time points (2 h and 6 h), there was no significant preferential accumulation of bismuth in any organ. At the 5-day time point, beyond the range of therapeutic interest, there was delayed excretion of bismuth from reticuloendothelial tissues relative to radioiodine from catabolized antibody. Excretion of catabolized DOTA-bismuth had an apparent t1/2 of approximately 1 day without the marked renal accumulation typical of the free bismuth ion. The compatibility of DOTA conjugation with antibody bioactivity and the stability of the radioactive bismuth complex in vivo provide important preclinical validation of the potential utility of this new chelating agent for 212Bi monoclonal antibody radioimmunotherapy in humans.

Tumor-associated GM-CSF overexpression induces immunoinhibitory molecules via STAT3 in myeloid-suppressor cells infiltrating liver metastases.

Assumptions that liver immune cells and immunosuppressive pathways are similar to their counterparts in other spaces have led to gaps in our understanding of intrahepatic neoplasm aggressiveness. Myeloid-derived suppressor cells (MDSCs) are potent inhibitors of antitumor immunity and pose a major obstacle to solid tumor treatment. Liver MDSCs (L-MDSCs) associated with liver metastases (LM) are particularly problematic by contributing to intrahepatic immunosuppression that promotes tumor progression. L-MDSCs have been reported to expand in response to granulocyte-macrophages colony-stimulating factor (GM-CSF) and suppress antitumor immunity in LM. To extend these findings, we examined mechanisms of intrahepatic immunosuppression exploited by L-MDSCs. We found that the majority of L-MDSCs co-expressed GM-CSF receptor (GM-CSF-R), indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PD-L1), while demonstrating high levels of signal transducer and activator of transcription factor 3 (STAT3) activation. GM-CSF-secreting tumor cells induced STAT3 phosphorylation in L-MDSCs in addition to expression of IDO and PD-L1. GM-CSF or GM-CSF-R blockade markedly reduced L-MDSC IDO and PD-L1 expression, implicating tumor-derived GM-CSF in supporting L-MDSC-immunoinhibitory molecule expression. Small-molecule inhibitors of Janus-activated kinase 2 (JAK2) and STAT3 also dramatically diminished IDO and PD-L1 expression in L-MDSCs. We determined that STAT3 exerts transcriptional control over L-MDSC IDO and PD-L1 expression by binding to the IDO1 and PD-L1 promoters. Our data suggest that the GM-CSF/JAK2/STAT3 axis in L-MDSCs drives immunosuppression in a model of LM and blockade of this pathway may enable rescue of intrahepatic antitumor immunity.

Regional CAR-T cell infusions for peritoneal carcinomatosis are superior to systemic delivery.

Metastatic spread of colorectal cancer (CRC) to the peritoneal cavity is common and difficult to treat, with many patients dying from malignant bowel obstruction. Chimeric antigen receptor T cell (CAR-T) immunotherapy has shown great promise, and we previously reported murine and phase I clinical studies on regional intrahepatic CAR-T infusion for CRC liver metastases. We are now studying intraperitoneal (IP) delivery of CAR-Ts for peritoneal carcinomatosis. Regional IP infusion of CAR-T resulted in superior protection against carcinoembryonic antigen (CEA+) peritoneal tumors, when compared with systemically infused CAR-Ts. IP CAR-Ts also provided prolonged protection against IP tumor re-challenges and demonstrated an increase in effector memory phenotype over time. IP CAR-Ts provided protection against tumor growth at distant subcutaneous (SC) sites in association with increases in serum IFNγ levels. Given the challenges posed by immunoinhibitory pathways in solid tumors, we combined IP CAR-T treatment with suppressor cell targeting. High frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) were found within the IP tumors, with MDSC expressing high levels of immunosuppressive PD-L1. Combinatorial IP CAR-T treatment with depleting antibodies against MDSC and Treg further improved efficacy against peritoneal metastases. Our data support further development of combinatorial IP CAR-T immunotherapy for peritoneal malignancies.

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.

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.

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.

Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors.

Immunoglobulin T-cell receptors (IgTCRs) combine the specificity of antibodies with the potency of cellular killing by grafting antibody recognition domains onto TCR signaling chains. IgTCR-modified T cells are thus redirected to kill tumor cells based on their expression of intact antigen on cell surfaces, bypassing the normal mechanism of activation through TCR-peptide-major histocompatibility complex (MHC) recognition. Melanoma is one of the most immunoresponsive of human cancers and has served as a prototype for the development of a number of immunotherapies. The target antigen for this study is the ganglioside GD3, which is highly expressed on metastatic melanoma with only minor immunologic cross-reaction with normal tissues. To determine an optimal configuration for therapy, four combinations of IgTCRs were prepared and studied: sFv-epsilon, sFv-zeta, Fab-epsilon, Fab-zeta. These were expressed on the surface of human T cells by retroviral transduction. IgTCR successfully redirected T-cell effectors in an MHC-unrestricted manner, in this case against a non-T-dependent antigen, with specific binding, activation, and cytotoxicity against GD3+ melanoma cells. Soluble GD3 in concentrations up to 100 microg/ml did not interfere with recognition and binding of membrane-bound antigen. Based on the outcomes of these structural and functional tests, the sFv-zeta construct was selected for clinical development. These results demonstrate key features that emphasize the potential of anti-GD3 IgTCR-modified autologous T cells for melanoma therapies.

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.

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.

High-dose intravenous gamma globulin to suppress alloimmune destruction of donor platelets.

We report the use of high-dose intravenous gamma globulin to overcome refractoriness to platelet transfusion in an alloimmunized patient with acute leukemia and thrombocytopenia. For two years the patient suffered recurrent gastrointestinal bleeding from an arteriovenous malformation and was given multiple transfusions, providing a basis for his allosensitization. Platelet counts had not increased following transfusions of random-donor or HLA-matched platelets. With intravenous gamma globulin, one hour after the transfusion of 9 to 15 units of platelets, the count increased by 30,000 to 90,000 and the half-life of transfused platelets increased to three to four hours from an estimated 0.05 hours prior to therapy. Intravenous gamma globulin arrested massive gastrointestinal bleeding and allowed the patient to undergo surgical resection of the small bowel with minimal operative blood loss.

Direct immunoprecipitation of antigen with phage displaying immunoglobulin fragment.

Phage libraries may display hormones, receptors, antibody fragments, etc., by fusion to phage envelope proteins. This report describes the direct precipitation of phage-Fab-antigen complexes by polyethylene glycol precipitation, resulting in highly selective and efficient recovery of antigen from complex mixtures without nonspecific protein contamination. The method demonstrates efficiency and specific recovery of phage-Fab-antigen complexes from a background of a complex mixture of unrelated proteins as may occur in the analysis of biological specimens. This simple, fast, and effective method allows isolation and characterization of target antigens, with no need to further process Fab or sFv, and may reasonably be extended to isolate any interacting partner molecule for any displayed protein.

An advanced solid support for immunoassays and other affinity applications.

A 6-mm diameter glass bead that has a hydrophilic coating containing epoxy groups has been developed for immunoassays and other affinity applications. These QuantAffinity beads covalently bind protein in the range of 200-400 ng per bead. Derivatization with protein in the presence of high concentrations of potassium phosphate (pH 7.0-8.0) generally gives complete coupling in 16 hours. No preactivation of the support or the protein is necessary. Low nonspecific binding and high retention of bound protein activity are inherent in the system due to the special surface chemistry. Several enzymatic and radioactive immunoassays as well as a biotin-avidin-based assay are described. A use of QuantAffinity beads in small-scale purification is also presented.