Robert L. Raison

Human cytokines suppress apoptosis of leukaemic CD5+ B cells and preserve expression of bcl-2.

Leukaemic CD5+ B cells obtained from B cell chronic lymphocytic leukaemia (B‐CLL) patients rapidly undergo apoptosis during in vitro culture. This is associated with down‐regulation in expression of bcl‐2. Spontaneous apoptosis of these cells contrasts their enhanced longevity in vivo and suggests that apoptosis‐inhibitory factors may be responsible for the accumulation of leukaemic cells in B‐CLL. The effect of different cytokines on apoptosis and bcl‐2 expression was examined in six populations of leukaemic CD5+ B cells. Consistent with previous data, IL‐4 and IFN‐γ suppressed apoptosis in 6/6 and 5/6 cell populations, respectively. Interestingly, the ability to suppress apoptosis in leukaemic CD5+ B cells was also found to be a property of IL‐2, IL‐6, IL‐13 and TNF‐α. In the presence of these cytokines, 10‐40% more viable cells were detected, compared with unstimulated cultures. Enhancement of cell viability and suppression of apoptosis were associated with a delay in down‐regulation of bcl‐2. These results suggest a role for autocrine and paracrine growth factors in the pathogenesis of B‐CLL, and indicate that cytokines which prevent apoptosis in vitro may be targets for treating this malignancy.

Antibody-antigen kinetics following immunization of rainbow trout (Oncorhynchus mykiss) with a T-cell dependent antigen.

Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout (Oncorhynchus mykiss) by measuring antibody-antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants (k(diss.)). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k(diss.)) was observed over the ensuing course of the immune response. An average k(diss.) of 3.5 x 10(-4)+/-0.27 x 10(-4)sec(-1) was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly (p<0.05). Further reduction in k(diss.) was observed, with a low of 1.2 x 10(-4)+/-0.06 x 10(-4)sec(-1) being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k(diss.) as the immune response progressed. While these data clearly demonstrate a 2-3 fold increase in antibody-antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.

Diverse binding site structures revealed in homology models of polyreactive immunoglobulins

We describe here computer-assisted homology models of the combiningsite structure of three polyreactive immunoglobulins. Template-based modelsof Fv (VL–VH) fragments were derived forthe surface IgM expressed by the malignant CD5 positive B cells from threepatients with chronic lymphocytic leukaemia (CLL). The conserved frameworkregions were constructed using crystal coordinates taken from highlyhomologous human variable domain structures (Pot and Hil). Complementaritydetermining regions (CDRs) were predicted by grafting loops, taken fromknown immunoglobulin structures, onto the Fv framework models. The CDRtemplates were chosen, where possible, to be of the same length and of highresidue identity or similarity. LCDR1, 2 and 3 as well as HCDR1 and 2 forthe Fv were constructed using this strategy. For HCDR3 prediction, adatabase containing the Cartesian coordinates of 30 of these loops wascompiled from unliganded antibody X-ray crystallographic structures and anHCDR3 of the same length as that of the B CLL Fv was selected as a template.In one case (Yar), the resulting HCDR3 model gave unfavourable interactionswhen incorporated into the Fv model. This HCDR3 was therefore modelled usingan alternative strategy of construction of the loop stems, using apreviously described HCDR3 conformation (Pot), followed by chain closurewith a β-turn. The template models were subjected to positionalrefinement using energy minimisation and molecular dynamics simulations(X-PLOR). An electrostatic surface description (GRASP) did not reveal acommon structural feature within the binding sites of the three polyreactiveFv. Thus, polyreactive immunoglobulins may recognise similar and multipleantigens through a diverse array of binding site structures.

Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin

BACKGROUND The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. OBJECTIVES We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. STUDY DESIGN The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). RESULTS Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. CONCLUSION The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.

Chemotactic responses of hagfish (Vertebrata, Agnatha) leucocytes

The chemotactic responses of hagfish leucocytes were tested using a variety of chemoattractants. Leucocyte migration was significantly enhanced by purified mammalian complement anaphylotoxin (C5a) and LPS-activated hagfish plasma. Checkerboard analyses confirmed that the responses of leucocytes to both of these chemoattractants were directed along concentration gradients (chemotaxis) and did not result from accelerated random movement (chemokinesis). Chemotaxis was undertaken by leucocyte fractions that were enriched in granulocytes, the predominant phagocytic cells of hagfish. The data suggest that chemotactic mechanisms may have been conserved during evolution to such a degree that mammalian chemoattractants can bind and activate chemotactic receptors on hagfish leucocytes. Moreover, hagfish appear to express plasma proteins that are structurally and functionally homologous to mammalian complement anaphylotoxins.

Standardization of antigen E in ragweed pollen extracts using a monoclonal antibody-based enzyme immunoassay☆

A hybridoma-derived monoclonal IgG antibody specific for ragweed AgE was used to develop a competitive binding enzyme immunoassay suitable for quantitation of antigen E levels in ragweed pollen extracts. The assay was capable of detecting as little as 30 ng/ml AgE in crude pollen extracts. The monoclonal antibody was shown to react with AgE present in commercial pooled pollen extracts from a number of ragweed species as well as a laboratory extract from a single species. In contrast to previous conventional xenoantisera, it could distinguish true ragweed (Ambrosia sp) from false ragweed (Franseria sp). The use of monoclonal antibodies in assay systems such as this offers a reproducible and widely applicable method for allergen standardization.

Quantitation of free κ light chains in serum and urine using a monoclonal antibody based inhibition enzyme-linked immunoassay

The murine monoclonal antibody K-1-21 with specificity for free but not heavy chain-associated kappa chains was used to develop an inhibition enzyme immunoassay for quantitation of free kappa chains in serum and urine. The assay utilizes a biotin conjugate of the monoclonal antibody and has an effective working concentration range of 0.5-50 micrograms/ml free kappa chain. Normal serum and urine yielded mean values of 1.2 and 4.9 micrograms/ml respectively using this assay system. Serum levels in patients with kappa chain myelomatosis ranged from 2 to 1700 micrograms/ml while serum from patients with lambda chain malignancies exhibited normal free kappa chain levels. The importance of the ability to readily quantitate light chains in serum was demonstrated by the finding in one patient of an absence of free kappa chains in the urine despite elevated levels of serum kappa chains. A significant drop in serum-free kappa chain levels was observed in this patient following a course of chemotherapy. The assay should prove valuable in the diagnosis, prognosis and monitoring of B cell neoplasias characterized by free kappa light chain secretion.

Single-step purification of immunoglobulin M on C1q-Sepharose

A rapid and simple affinity chromatography method for purifying IgM from myeloma serum and ascites fluid is described. Complement protein C1q is coupled to Sepharose with an efficiency of 35%, giving 1.7 mg of C1q bound/ml of gel. This C1q-Sepharose selectively binds IgM from crude samples at 5 degrees C, with a capacity of 0.4 mg of IgM/ml of gel. The bound IgM may be eluted simply and isocratically by bringing the gel to room temperature for 2 h, or by washing with buffer containing 0.5 M KI. The eluted IgM is highly pure by SDS-PAGE and double immunodiffusion analysis, although IgG may be a potential contaminant. The C1q-Sepharose is stable for at least 18 months.

Interleukin-10 Inhibits the In vitro Proliferation of Human Activated Leukemic CD5+ B-Cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterised by the proliferation and accumulation of sIgM+/CD5+ B-cells that fail to progress to the final stages of B-cell development. Despite their developmental arrest, leukemic CD5+ B-cells can undergo proliferation in vitro in the presence of different activators including phorbol esters, antibodies to cell surface antigens and human cytokines. Interleukin-10 (IL-10) has recently been found to inhibit CLL B-cell function in vitro by inducing apoptosis and down-regulating expression of bcl-2. Here, we examined the effect of IL-10 on proliferation, RNA synthesis, immunoglobulin (IgM) secretion and viability of leukemic CD5+ B-cells induced by activation with the phorbol ester PMA, alone or in combination with anti-Ig. IL-10 reduced PMA and PMA/anti-Ig induced proliferation and RNA synthesis by 50-80% and 15-40% respectively. Although proliferation and RNA synthesis induced by PMA/anti-Ig could be enhanced by the addition of IL-2, IL-4, IL-13, IFN-gamma or TNF-alpha, the presence of these cytokines failed to abrogate the IL-10-mediated inhibition of leukemic CD5+ B-cell activation. In contrast to the effects on proliferation and RNA synthesis, IL-10 did not inhibit IgM secretion, and had only a minimal effect on the viability of activated cells. Our results indicate that IL-10 inhibits proliferation of leukemic CD5+ B-cells by a mechanism distinct from induction of apoptosis and support the proposal for the utilisation of IL-10 in the therapy of B-CLL.

A new microsphere-based immunofluorescence assay for antibodies to membrane-associated antigens

The screening of panels of hybridoma supernatants for specific secreted monoclonal antibodies is often achieved by cellular immunofluorescent staining and flow cytometric analysis. In some circumstances such assays are difficult because the required antigen-bearing cell population is not suitable for use in flow cytometry, has limited cell cycle expression or poor in vitro growth. A method is presented here that provides a solution to these difficulties. A system was developed, using polyacrylamide microspheres coupled with cell membranes, which permitted the production of an easily stored, standardised antigen source which could be used in subsequent flow cytometric assays. Studies comparing the binding of antibodies to whole cells and cell membrane-coupled microspheres indicate a strong qualitative and quantitative correlation in the expression of surface antigens. It is shown here that membrane antigens can be stored coupled to microspheres for months and still retain good reactivity with the appropriate antibodies. This same technique could be used in studies of antigens other than those of the mammalian cell membrane - for example, membrane antigens of sub-cellular organelles such as the mitochondrion and endoplasmic reticulum, plant or bacterial membrane antigens, or antigens not associated with membranes such as viral proteins.