Biomedical subjects

N A Norris

Publications and source records attributed to N A Norris.

11 recordsLinked to original sources

PubMed1996

Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments.

Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2-3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly-G tailing the first strand cDNA followed by anchor PCR with a forward poly-C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL-link-VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-CH2-CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide > or = tag > or = or in a tail-less form. Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.

Amino Acid Sequence↗

PubMed1996

Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen.

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.

Amino Acid Sequence↗

PubMed1995

Activity of a single-chain immunotoxin that selectively kills lymphoma and other B-lineage cells expressing the CD40 antigen.

We have constructed anti-CD40 immunotoxins consisting of the single chain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a truncated form of Pseudomonas exotoxin, PE40. CD40 is an integral membrane glycoprotein found on the surface of B-lineage cells, including lymphomas and leukemias, as well as certain carcinomas. Two forms of the immunotoxin were constructed, one with the light chain variable (VL) region of the sFv preceding the heavy chain variable region (VH) [G28-5 sFv(VL-VH)-PE40] and the second with the sFv in the opposite orientation [G28-5 sFv(VH-VL)-PE40]. Although both forms of G28-5 sFv-PE40 specifically bound to CD40 in ELISAs, the binding of G28-5 sFv(VL-VH)-PE40 was > 10-fold higher. A number of malignant B- and T-cell lines were screened for CD40 expression and susceptibility to G28-5 sFv(VL-VH)-PE40. All of the B-lineage cells tested were CD40 positive and sensitive to the anti-CD40 immunotoxin, with EC50s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins. Consistent with the antigen-binding results, the VL-VH immunotoxin was > 10-fold more cytotoxic than the VH-VL immunotoxin. The anti-CD40 single-chain immunotoxin fusion protein G28-5 sFv(VL-VH)-PE40 represents a potent and specific cytotoxic agent for the elimination of normal and transformed B-lineage cells expressing CD40.

ADP Ribose Transferases↗

PubMed1994

Single-chain mono- and bispecific antibody derivatives with novel biological properties and antitumour activity from a COS cell transient expression system.

Single-chain antibody molecules were expressed from modified eukaryotic expression vectors as individual protein domains encoded on interchangeable cDNA cassettes. Two different single-chain antibody derivatives were constructed by linking individual light- and heavy-chain variable domains. The first was specific for the L6 tumour-associated antigen and the second was specific for human CD3. Each single-chain variable domain was genetically fused with an Fc 'tag' and expressed as a fusion protein in a COS cell transient transfection system. These single-chain antibody derivatives demonstrated specific binding to cells expressing appropriate antigen and bound with affinities similar to native antibody. The CD3 single chain molecule mediated stronger activation of PLC gamma 1 and similar levels of T-cell proliferation compared with native antibody. A bispecific Fv single-chain cassette was created by fusing the expression cassettes encoding the binding domains for L6 and CD3 single-chain molecules using oligonucleotide primers encoding a short 27-residue 'helical' peptide linker. The CD3-L6 variable domains were fused to the Fc tag and expressed in COS cells. The CD3-L6FvIg bispecific fusion protein mediated adhesion between T cells and L6-positive tumour cells, and stimulated potent T-cell proliferation and cytotoxicity against tumour cells expressing the L6 antigen.

Amino Acid Sequence↗

PubMed1992

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.

Binding Sites↗

PubMed1991

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.

Animals↗

PubMed1991

Signal transduction by HLA-DR is mediated by tyrosine kinase(s) and regulated by CD45 in activated T cells.

Recently, it was shown that HLA class II molecules on B cells and activated human T cells can transmit signals involving tyrosine phosphorylation of specific proteins, activation of the inositol phospholipid pathway, and release of cytosolic free Ca2+(Ca2+)i. The regulation of class II induced signals is poorly understood, however, and it remained unknown whether these pathways were coupled or activated independently. Here we show that a specific inhibitor of protein tyrosine kinases (PTK), herbimycin, abrogated DR-induced elevation of (Ca2+)i in activated human T cells. Genistein, belonging to another family of PTK inhibitors, had weaker but significant inhibitory effects on DR-induced (Ca2+)i responses. CD45 crosslinking with DR almost completely abrogated DR-induced (Ca2+)i responses and profoundly changed the PTK profiles. In contrast, CD4 crosslinking with DR enhanced the (Ca2+)i responses, but the inhibitory effect of CD45 dominated over the enhancing effect of CD4. These data indicate that PTK activation is obligatory for DR-induced (Ca2+)i responses, suggesting a linkage between these pathways in class II signal transduction. This conclusion is consistent with our observation that in activated human T cells, class II signals are up regulated by CD4, which is associated with p56lck, and down regulated by CD45, which is a tyrosine phosphatase.

Antigens, CD↗

PubMed1989

Enhanced transmembrane signalling activity of monoclonal antibody heteroconjugates suggests molecular interactions between receptors on the T cell surface.

Signal transduction occurs through multiple receptors expressed on mature, resting T cells. In addition to the CD3-T cell receptor complex, the CD2, CD4, CD5, CD7, CD8 and CD28 receptors mobilize cytoplasmic calcium within minutes of binding with monoclonal antibodies and additional crosslinking occurs on the cell surface. As an approach to study the interactions between these receptors and their transduced signals, monoclonal antibodies to each of these receptors were covalently coupled as heteroconjugates and investigated for activity in cytoplasmic calcium mobilization using indo-1 and flow cytometry. Of a total of 35 conjugates studied, there were seven heteroconjugates that showed an increase in activity and these consisted of either certain conjugates of anti-CD3 or certain conjugates of anti-CD5. The CD3-CD2, CD3-CD4, CD3-CD6 and CD3-CD8 heteroconjugates each gained two to three orders of magnitude in titer in calcium mobilization compared to unconjugated CD3 or the CD3-CD3 conjugate. The increase in activity was not accompanied by an increase in binding titer, indicating that signal transduction occurred at lower levels of receptor occupancy. The increased activity was dependent in each case on the relevant second receptor, since unconjugated CD2, CD4, CD6 or CD8 MAb could block the activity of the corresponding heteroconjugate. Neither CD3-CD5, CD3-CD28 or CD3-CD3 conjugates gained activity, whereas CD3-CD7 heteroconjugates gained slightly in activity. The heteroconjugates with CD5 that acquired ability to mobilize calcium at low concns (less than 5 micrograms/ml) were CD5-CD4, CD5-CD8 and CD5-CD6. Their activity could be inhibited by either CD5 MAb or the second MAb of the heteroconjugate. The increased activity of CD3 or CD5 heteroconjugates was observed in the absence of extracellular calcium. Size exclusion chromatography of heteroconjugates demonstrated that 1:1 ratios were optimal, but larger conjugates were also active. These results suggest that certain receptors are capable for molecular interactions on the cell surface to form complexes with enhanced activity in signal transduction leading to calcium mobilization.

Animals↗

PubMed1989

Signal transduction in lymphocyte activation through crosslinking of HLA class I molecules.

The inhibitory effect of anti-HLA class I monoclonal antibodies on lymphocyte proliferation has been well documented. However, recent data suggest that anti-HLA class I monoclonal antibodies can enhance lymphocyte proliferation via both anti-CD3-induced (1,2) and anti-CD2-induced (3) activation pathways. Here we demonstrate that both inhibition and activation can be regulated by the degree of aggregation of HLA class I antigens. Crosslinking of monoclonal antibodies specific for HLA-A, HLA-B, or monomorphic determinants (using anti-IgG2 and/or anti-Ig kappa "second step" monoclonal antibodies) increased the capacity of anti-HLA class I monoclonal antibodies to inhibit phytohemagglutinin-induced proliferation. However, the cytosolic free calcium concentration was increased in CD4+ cells, CD8+ cells, B cells, and CD16+ cells when anti-HLA class I monoclonal antibodies were crosslinked, suggesting that an activation signal was generated by aggregation of the corresponding antigens. Indeed, inositol 1,4,5-trisphosphate could be detected in peripheral blood lymphocytes following crosslinking of anti-HLA class I monoclonal antibodies. Class I aggregation also induced proliferation of peripheral blood mononuclear cells in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Strong conditions of crosslinking (monomorphic monoclonal antibody plus both anti-IgG2 and anti-Ig kappa) induced CD25 expression and responsiveness to recombinant interleukin 2. Our results suggest that aggregation of HLA class I antigens primed cells to become activated in the presence of progression signals including phorbol 12-myristate 13-acetate, recombinant interleukin 2, or anti-CD5 plus anti-CD28 monoclonal antibodies.

Antibodies, Monoclonal↗

PubMed1988

Antibody to a novel 95-kDa surface glycoprotein on human B cells induces calcium mobilization and B cell activation.

These findings characterize a 95-kDa glycoprotein on the surface of B lymphocytes recognized by the mAb G28-8. This protein (designated Bgp95), previously classified as a CD39 molecule, is unique based on functional, cell distribution, and immunochemical criteria. Biochemical analyses revealed that Bgp95 is a 95-kDa glycoprotein with N-linked carbohydrate and is reduced to about 70-kDa after treatment with endoglycopeptidase F. In functional studies, stimulation by G28-8 mAb or its F(ab')2 fragments induced a G0 to G1 cell cycle transition and was synergistic with PMA, anti-mu, or anti-CDw40 in stimulating proliferation of resting B cells. G28-8 mAb also could induce increases of cytoplasmic free calcium concentration [Ca2+]i in a subpopulation of tonsillar or peripheral blood B cells. The G28-8 mAb alone induced a steady increase in [Ca2+]i detectable even 1 h after stimulation. Cross-linking the G28-8 mAb with a second mAb specific for murine kappa light chains induced a more rapid increase of [Ca2+]i which peaked at 10 to 20 min and then declined. At 1 h after stimulation, [Ca2+]i was higher in B cells stimulated with G28-8 alone than in B cells stimulated with G28-8 plus anti-kappa. The same conditions of cross-linking with the anti-kappa which increased the kinetics of the [Ca2+]i response decreased the proliferative response which otherwise followed co-incubation of the mAb with B cell growth factor or PMA. Thus, conditions leading to rapid but transient [Ca2+]i increase via Bgp95 may not be as effective at stimulating B cell proliferation as conditions favoring a slower prolonged [Ca2+]i response. Although the Bgp95 molecule is present on activated buoyant tonsillar B cells, mAb to Bgp95 did not trigger [Ca2+]i increases in these cells. These results suggest that the Bgp95 protein may function in early B cell activation and that its signal mechanisms are altered by the activation state of the cell.

Antibodies, Monoclonal↗

PubMed1987

An immunoglobulin light chain dimer with CD4 antigen specificity.

A subclone that had lost its IgG1 heavy chain was derived from a hybridoma cell line G17-2 that produces an anti-CD4 monoclonal antibody. This subclone was found to secrete a kappa light chain dimer (LCD) that could bind to the CD4 antigen expressed on a subset of human T lymphocytes. The light chain dimer bound to the same or similar epitope as the parental antibody since it blocked the binding of the parenteral anti-CD4 MAb but not the binding of another anti-CD4 MAb G19-2 that recognizes a different epitope. A rabbit anti-idiotype prepared against G17-2 crossreacted with the LCD and could block the antigen binding of both G17-2 and the LCD. Scatchard analysis performed with 35S-methionine or 3H-leucine labelled LCD showed an association constant Ka of 2.2 x 10(7) M-1, whereas the G17-2 parental antibody showed an association constant Ka of 2.5 x 10(9) M-1. These data indicate that the antigen specificity of the G17-2 parental MAb is conferred to a large extent by its light chain. The LCD was expressed on the surface of the LCD-producing hybridoma cells. Southern blot analysis with C kappa and J kappa probes demonstrated a single kappa transcription units which does not have any unusual DNA rearrangements and is distinct from the NS-1 kappa genes. To our knowledge, this LCD is unique in its ability to bind to a large (55,000 mol. wt) glycoprotein antigen.

Animals↗