A quantitative immunoassay utilizing Escherichia coli cells possessing surface-expressed single chain Fv molecules

A facile, quantitative immunoassay is described that utilizes Escherichia coli (E. coli) bacteria expressing single chain Fv (scFv) antibody fragments attached to the cell surface. A Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity (Kd = 2.2 x 10(-9) M) and that there are 60,000 scFv molecules per cell. The cells are used as the antibody reagent in the assay, and, following incubation with analyte, simple centrifugation is used to separate the antibody-bound from unbound analyte. The immunoassay is rapid and accurate down to the nanomolar level. In addition, a variety of detection strategies can be used, and the immunoassay is not adversely affected by the presence of animal serum. A key advantage of the new immunoassay is that the antibody reagent can be inexpensively produced in a "ready to use" form by simply growing cultures of the bacteria.     

Lovastatin inhibits T-cell antigen receptor signaling independent of its effects on ras

Lovastatin, a cholesterol-lowering drug, has antiproliferative properties that may be related to its inhibition of protein isoprenylation. We examined the effects of lovastatin on signal transduction via the T-cell antigen receptor (TCR). Lovastatin inhibited both proximal and distal TCR-mediated signaling events in a time- and concentration-dependent manner in the human Jurkat T-cell line. Upregulation of CD69 surface expression after TCR stimulation was blocked by lovastatin, although no inhibition of phorbol ester-induced CD69 expression was noted. Proximal TCR-mediated signaling events, including intracellular calcium mobilization, inositol phosphate production, and tyrosine phosphorylation of phospholipase Cgamma1, were similarly inhibited by lovastatin, although global protein tyrosine kinase activity remained intact. In a Jurkat variant transfected with the human type-1 muscarinic receptor, lovastatin also inhibited TCR-mediated calcium mobilization and inositol phosphate production but failed to affect muscarinic receptor-induced responses. Lovastatin, at similar doses, also disrupted post-translational processing of ras and inhibited ras-dependent signals, including phosphorylation and activation of mitogen-associated protein kinase after TCR stimulation. These findings suggest that the antiproliferative properties of lovastatin may be independent of ras and could result from uncoupling protein tyrosine kinases from distinct signal transduction pathways.     

Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease A

The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M(-1) in PBS at pH 7.2 and 37 degrees C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.     

Biased T-cell antigen receptor repertoire in Lyme arthritis

A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete, Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation with B. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) beta-chain variable (Vbeta) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vbeta2 and Vbeta6 in the latter. This is remarkably similar to our previous findings in studies of rheumatoid arthritis and to other reports on psoriatic skin lesions. However, stimulation of synovial fluid T cells with B. burgdorferi provoked active proliferation but not a statistically significant increase in expression of any TCR Vbeta, including Vbeta2 and Vbeta6. Collectively, the findings suggest that the skewing of the TCR repertoire of fresh synovial fluid in Lyme arthritis may represent more a synovium-tropic or nonspecific inflammatory response, similar to that occurring in rheumatoid arthritis or psoriasis, rather than a specific Borrelia reaction.     

CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.     

DNA vaccines against lymphoma: promotion of anti-idiotypic antibody responses induced by single chain Fv genes by fusion to tetanus toxin fragment C

Idiotypic determinants can act as tumor-associated Ags for B cell lymphoma. Vaccination with idiotypic protein and adjuvant is known to induce specific protection against lymphoma challenge in mice, largely mediated by anti-idiotypic Ab. For facilitating the approach for patients, the V(H) and V(L) genes used to encode the individual idiotypic determinants of each tumor can be obtained by PCR and assembled as single chain Fv (scFv). DNA vaccines containing scFv sequences alone induce low and poorly reproducible levels of anti-idiotypic Ab, likely to be insufficient to suppress tumor in patients. In addition, it may be necessary to break tolerance to Id in tumor bearers. By fusing the gene for fragment C of tetanus toxin to the C terminus of human scFv, we have promoted the anti-scFv Ab response in mice by >50-fold in three of three cases. The induced Abs are mainly against idiotypic determinants, and react specifically with patients' tumor cells, indicating optimal folding of the scFv molecule in the fusion protein. For both antigenic components of the DNA vaccine, the IgG subclass distribution showed a relative increase in IgG2a as compared with vaccination with IgM protein in adjuvant. In patients, the fusion gene should both promote anti-idiotypic Ab and induce Abs against fragment C of tetanus toxin. The latter response would provide a potentially useful comparative measure of the ability of patients to respond to conventional Ag delivered via DNA.     

Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules

A plasmid vector was constructed for the expression of a single chain Fv domain of mouse mAb to Z-DNA (antibody Z22), which is encoded by VH10 and V kappa 10 gene family members along with Dsp2, JH4, and J kappa 4 segments. The vector coded for a PhoA secretion signal, VH segment, flexible peptide linker, VL segment, (His)5, and a protein A domain. Unique restriction sites allowed exchange of the segments as cassettes. Bacteria transformed with the vector secreted soluble recombinant Fv with specific Z-DNA-binding activity. When the L chain of Z22 was replaced with a library of splenic VL cDNA from a mouse immunized with Z-DNA, only a light chain closely resembling that of the original Z22 (differing at six amino acid positions) yielded Fv with Z-DNA-binding activity. The Fv with this L chain replacement had a lowered affinity, but remained selective for Z-DNA. Replacement of the Z22 H chain with a mixture of 11 VH10-encoded H chains yielded two Z-DNA binding clones, but they bound B-DNA and denatured DNA as well as Z-DNA. The replacement clones indicate the importance of the H chain CDR3 and particular VH-VL combinations in formation of specific antibodies to Z-DNA.     

Immune responses in patients with T-cell lymphoma treated with an anti-idiotype antibody mimicking a highly restricted T-cell antigen

We generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) to a highly restricted T-cell antigen designated glycoprotein (gp) 37 that is found on T-cell malignancies but not on normal cells. gp37 is identified by the murine monoclonal antibody SN2 (Ab1) against which the Ab2 was raised. Each of four patients with T-cell lymphoma predominantly confined to the skin received a minimum of four intracutaneous injections of aluminum hydroxide precipitated anti-idiotype murine monoclonal antibody (1 mg/injection) given every 2 weeks. For responding patients, injections were continued on a monthly basis. All tumors were measured along orthogonal major and minor axes, using a ruler and/or calipers, by the same observer. Tumor sizes were documented photographically. Three of the four patients developed specific idiotypic humoral immune responses, and two of the four patients also demonstrated idiotypic cell-mediated responses. Humoral responses included binding of the patients' sera to the anti-idiotype antibody as well as specific inhibition of binding of the SN2 antibody (Ab1) to the anti-idiotype antibody (Ab2). Anti-anti-idiotypic (Ab3) antibody from one patient's serum bound specifically to the gp37-positive cell line MOLT-4 and also to semipurified gp37 antigen. Cell-mediated responses were demonstrated by specific proliferative response to the aluminum hydroxide precipitated anti-idiotype antibody by patients' peripheral blood mononuclear cells. While three of the four patients had extensive disease and did not have clinical responses, one of the patients who had nine discrete skin tumors and peripheral blood involvement without other detectable disease had virtually complete disappearance of the tumors lasting over 11 months. Our results demonstrate that this particular anti-idiotype antibody can induce humoral and cellular immune responses, and at least in one patient led to a meaningful therapeutic response. Future trials should focus on immunocompetent patients with minimal disease.     

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.     

A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function

Ras and Raf-1 are key proteins involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Raf-1 functions downstream of Ras in many signaling pathways. Although Raf-1 directly associates with GTP-bound Ras, an effect of this interaction on Raf-1 activity in vivo has not been established. To examine the biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In this report, we show that a single amino acid mutation in Raf-1 (Arg89 to Leu) disrupted the interaction with Ras in vitro and in the yeast two-hybrid system. This mutation prevented Ras-mediated but not tyrosine kinase-mediated enzymatic activation of Raf-1 in the baculovirus/Sf9 expression system. Furthermore, kinase-defective Raf-1 proteins containing the Arg89-->Leu mutation were no longer dominant-inhibitory or capable of blocking Ras-mediated signal transduction in Xenopus laevis oocytes. These results demonstrate that the association of Raf-1 and Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg89 as a critical residue involved in this interaction. In addition, the finding that tyrosine kinases can stimulate the enzymatic activity of Raf-1 proteins containing a mutation at the Ras-interaction site suggests that Raf-1 can be activated by Ras-independent pathways.     

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.     

Induction of late activation events by Igbeta signaling subunit of B-cell receptor in Jurkat T-cell without tyrosine phosphorylation

T-lymphocyte activation consists of multiple intracellular signaling events, eventually leading to cellular proliferation by the control of cytokine gene expression and the acquisition of diverse effector function. To investigate the functional specificity of ITAM (Immunoreceptor Tyrosine-based Activation Motif), chimeric molecules CD8-zeta, CD8-Igalpha, CD8-Igbeta, which contain the extracellular and transmembrane domains of the human CD8alpha molecule and the cytoplasmic tail of T-cell receptor (TcR) chain, Igalpha or Igbeta subunit of B-cell receptor, respectively, were stably expressed in a Jurkat cell line. Upon stimulation with anti-CD8 mAb OKT8, CD8-zeta and CD8-Igalpha chimeric proteins induced tyrosine phosphorylation of various cytoplasmic substrates as seen in TcR stimulation. They were also capable of stimulating IL-2 gene expression in a NF-AT dependent manner and inducing CD69 expression on the surface. However, stimulation of CD8-Igbeta can induce activation of CD69 surface expression and IL-2 gene expression equivalent to the level by CD8-Igalpha and CD8-zeta without induction of the tyrosine phosphorylation of intracellular signaling molecules. These results suggested that some of signaling chains containing ITAM may utilize a signal pathway without substrate tyrosine phosphorylation during T-cell activation leading to the IL-2 secretion.     

Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library

OBJECTIVES: The authors examined how the courts have responded to public and private insurers' use of medical appropriateness criteria to establish coverage and payment policies. METHODS: A structured review of all federal and state court health insurance cases decided between 1960 and June 1994 that involved a dispute involving medical appropriateness was performed. A total of 3,215 published court decisions were analyzed, of which 203 met the criteria of relevance and 124 explicitly mentioned medical appropriateness criteria. The main outcome variable was whether the court ordered the insurer to provide coverage. RESULTS: In 185 cases, a definitive decision was rendered, and the insurer was required to pay in 57% of the decisions. Whether the insurer relied on an assessment or not, whether the assessment process was formal or informal, and who conducted the assessment did not appear to influence courts' decisions, nor did the specificity of the coverage exclusion. Significant predictors of courts ordering coverage were court jurisdiction, contract language assigning discretion to the insurer, severity of patient's condition, and whether the treatment appeared to work for the particular patient. CONCLUSIONS: For practice guidelines to be accepted by the courts, it is more important to focus on how insurance contracts are written than on how medical assessments are performed.     

CEP-751 inhibits TRK receptor tyrosine kinase activity in vitro exhibits anti-tumor activity

The present report describes the in vitro and in vivo profile of CEP-751, a novel receptor tyrosine kinase inhibitor. CEP-751 at 100 nM inhibits the receptor tyrosine kinase activity of the neurotrophin receptors trkA, trkB and trkC. CEP-751 has no effect on activity of receptors for EGF, IGF-I, insulin or on erbB2; inhibition of receptors for PDGF and bFGF was observed but occurred with lesser potency than inhibition of trk. CEP-751 exhibited anti-tumor efficacy against tumors derived from NIH3T3 cells transfected with trkA. Inhibition of trk phosphorylation could also be measured in these tumors, suggesting that anti-tumor efficacy of CEP-751 is related to inhibition of trk receptor tyrosine kinase activity. CEP-751 was found to be without effect when administered to nude mice bearing SK-OV-3 tumors, which overexpress erbB2 receptors, providing further evidence that inhibition of tumor growth may be related to inhibition of trk receptor tyrosine kinase activity. Our data indicate that CEP-751 is a potent trk inhibitor which possesses anti-tumor activity.     

Sodium saccharin inhibits adenylyl cyclase activity in non-taste cells

We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5-50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5-50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg(2+)-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn(2+)-ATP and GDP beta S, a stable GDP analogue. The effect of NaSacch was not mediated through either the A1-adenosine receptor (A1R) or the alpha 2-adrenergic receptor (alpha 2AR). The inhibitory effect of NaSacch was additive to that of A1R agonist and was not blocked by the addition of the alpha 2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined.     

Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain

Interleukin-13 (IL-13) is a cytokine produced primarily by activated T lymphocytes. It exerts a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes. The effects of IL-13 on target cells are often similar to the effects of IL-4, which is another cytokine product of activated T lymphocytes. We recently described the expression of intermediate- to high-affinity receptors for IL-13 (IL-13R) on renal cell carcinoma (RCC) cells. In the present study, we examined the effect of IL-13 on the growth of RCC cells as measured by [3H]thymidine uptake and a clonogenic assay. In addition, we used an IL-4R-specific antibody to examine the specificity of IL-4R and IL-13R binding and function. We observed that IL-13 inhibited RCC cell proliferation by up to 50% and colony formation by up to 32% when compared with cells cultured in medium alone. A combination of IL-4 and IL-13 did not have an additive or synergistic effect on the growth of RCC cells. These cells expressed mRNA for IL-13 and secreted immunoreactive IL-13 protein in culture. The growth-inhibitory effects of IL-13 were specific, because they were not affected by antibodies to IL-4 or to the 140-kilodalton subunit of IL-4R. Furthermore, polyclonal antibodies to IL-4R failed to inhibit the binding of 125I-IL-13 to RCC cells. These results indicate that IL-13 has significant antiproliferative effects on human RCC cells, and the inhibition of IL-13 effects by anti-IL-4R antibody previously reported in lymphoid cells does not occur in RCC cells.