César Milstein. Extracts from an interview with David Secher in 1999 / César Milstein: Extractos de una entrevista con el autor en 1999

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Pharmacokinetics and immune response of 131I-chimeric mouse/human B72.3 (human gamma 4) monoclonal antibody in humans.

Chimeric B72.3, composed of the V-regions of murine B72.3 and the constant regions of human immunoglobulin G4 heavy and kappa light chain, was administered as a 131I-labeled conjugate to 12 patients with metastatic colon cancer. Seven of these patients had an antibody response after initial infusion, and the immune response was primarily directed to the murine V-region, although a small proportion of the antibody response was directed to topographical epitopes requiring the presence of both murine V-region and human CH-1 and kappa constant regions (neo-epitopes). The pharmacokinetics included a plasma disappearance curve best fit by a two-compartmental model with an alpha t 1/2 of 18 +/- 7 h and a beta t 1/2 of 224 +/- 66 h. A second infusion of the same dose of 131I-chimeric B72.3 was administered to four of these patients 8 wk after the first infusion. Two patients who had a high antibody response to initial infusion had an anamnestic antibody response, and the infused ch-B72.3 rapidly disappeared from the circulation with associated immune complexes and free 131I in the plasma. One patient with no initial antibody response had no antibody response and identical pharmacokinetics on second infusion. One patient with a modest transient antibody response to initial infusion had no antibody response on second infusion and a modest shortening of plasma circulation. Thus, the human immunoglobulin G4 isotype chimeric B72.3 monoclonal antibody has a plasma half-life 6 to 8 times as long as murine B72.3 and retains considerable immunogenicity in some patients which can adversely affect repetitive infusions.

From laboratory to clinic: the development of an immunological reagent.

Access to a wide range of high quality and increasingly sophisticated reagents and equipment has underpinned the great surge of knowledge in basic immunology and the growing interest in clinical immunointervention. In this article, the first in an occasional series on immunological research and development in industry, Sue Bright and colleagues outline the key steps in a development programme to take a humanized monoclonal antibody into the clinic. The procedures involved in developing such reagents, particularly for clinical use, are long and require considerable ingenuity and scientific creativity.

Treatment of advanced condylomata acuminata with semi-purified and purified human leukocyte interferon.

Twelve patients with advanced condylomata acuminata were treated by systemic human leukocyte interferon (IFN) therapy. Semi-purified and purified preparations were both able to affect condylomatous growth. Treatment of the patients at various periods of their disease resulted in one complete remission, 6 partial remissions, 4 minimal responses while one case showed progressive disease. Side-effects were unexpectedly common in these advanced patients and 4 of them had to stop IFN treatment.

Identification of a monoclonal antibody to abscission tissue that recognises xylose/fucose-containing N-linked oligosaccharides from higher plants.

Monoclonal antibodies raised against extracts of the rachis abscission zone of Sambucus nigra L. were selected for high reactivity towards abscission-zone proteins. One antibody (YZ1/2.23) has been shown to cross-react, by both indirect and competition enzyme-linked immunosorbent assay and by Western blotting, with a number of plant enzymes including horseradish peroxidase, rice α-glucosidase, almond ß-glucosidase and the lectins from Phaseolus vulgaris and Erythrina cristagalli.The major N-linked oligosaccharide isolated from horseradish peroxidase has the sequence Manα 3(Manα6)(Xylß2)Manß4GlcNAcß4(Fucα3) GlcNAc. This oligosaccharide was found to be a potent inhibitor of the binding of YZ1/2.23 to the intact glycoprotein. The common determinant is therefore contained within this structure.

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice.

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.

The regulation of membrane-bound and secreted immunoglobulins in the human lymphoid cell line LICR-LON and human hybridomas.

The synthesis of membrane-bound and secreted immunoglobulin was investigated in the human line LICR-LON-HMy2, a cell line often used for the derivation of human hybridomas. PAGE-SDS analysis of immunoprecipitates obtained from 35S-methionine labelled cell lysates shows that LICR-LON synthesize a hitherto undetected membrane form of IgG (with a heavy chain of mol. wt 62,000) in addition to the secretor form of IgG already described (55,000 heavy chain). Tunicamycin treatment, pulse-chase experiments and Western blot analysis showed that both chains are synthesized as independent proteins. Hybridomas obtained after fusion of LICR-LON and human peripheral blood lymphocytes retained the ability of the parental cell line to synthesize gamma m and gamma s. Some of these hybrids synthesize and secrete IgM which presumably originates from the parental B-lymphocytes. Precipitation and PAGE-SDS analysis of membrane proteins after iodination of intact cells revealed only one heavy chain band, corresponding in size to that of the gamma m. No indication of the synthesis of the membrane form of IgM was found in the hybrids. These data show that the parental (lymphoid) phenotype (m and s-IgG) is codominant with the more differentiated phenotype (s-IgM) of the fusion partner cell (plasma cell). These observations are compatible with a class-specific m-s regulation operating on a different chromatin structure at the expressed Ig loci of each parental chromosome.

A clue to the basic defect in cystic fibrosis from cloning the CF antigen gene.

The metabolic basis of the autosomal recessive disease cystic fibrosis (CF) remains unidentified. Elevated levels of a serum protein in CF homozygotes and obligate heterozygotes have been described. As heterozygotes are clinically unaffected, any consistently observed abnormality in these individuals is a likely pointer to the aetiology of the disease. The gene for this serum protein, called cystic fibrosis (CF) antigen, has been mapped to chromosome 1. It is not the gene that is mutant in CF because this has since been assigned to chromosome 7 by cosegregation of the disease with closely linked DNA markers in CF families. CF antigen is a product of normal and leukaemic granulocytes and is inducible in the promyelocytic cell line HL60 (M.N., J.D., C. Hayward, F. Northrop, D.J.H.B., J. Walker, V. van H. and D.S.S., manuscript in preparation). We have isolated cDNA clones for this protein from a library constructed with messenger RNA from chronic myeloid leukaemia (CML) cells. The complete nucleotide sequence was obtained from the cDNA clone and by primer extension of mRNA. We have confirmed that the gene encoding CF antigen is on chromosome 1 and have localized it to a particular region. RNA blot analysis shows a 550-bp major transcript in CML cells and in induced HL60. The amino-acid sequence predicted from the nucleotide sequence shows significant homology with intestinal and brain calcium-binding proteins. Abnormal accumulation of such a protein in CF is a clue which must be pursued now that evidence is gathering that the basic defect in CF is in pathways controlling chloride channel activity.

Hairy cells possess more interferon receptors than other lymphoid cell types.

To investigate the possible direct effects of alpha-interferon (IFN-alpha) in hairy cell leukemia, IFN-alpha receptor expression by hairy cells (11 cases) was measured by a radiolabeling technique and compared with that of MOLT-4, chronic lymphocytic leukemia (15 cases), and various other leukemic and normal cell types. Purified peripheral blood and splenic hairy cells showed higher levels of receptor expression (approximately 1,000 +/- 200 binding sites/cell; 11 cases tested) than other normal and leukemic cell types. B cells from normal blood and tonsils showed low levels of receptors (approximately 120 +/- 100 binding sites/cell), while a range of B cell leukemias displayed intermediate levels of expression (100-500 sites/cell). In the 15 cases of chronic lymphocytic leukemia tested, 530 +/- 330 binding sites/cell) were demonstrated, the high standard deviation reflecting the fact that one third of cases had receptor levels comparable with those in hairy cell leukemia. Normal and hairy cell leukemia T cells, red cells, and platelets had no demonstrable IFN-receptors. These findings may be relevant to the efficacy of IFN in hairy cell leukemia.

Effect of purified human interferon-alpha on the expression of differentiation antigens and mitogen reactivity of cultured human thymic cells.

Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-alpha. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA+ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses--but not interleukin 2 responses--were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.

The mechanism of action of interferon-alpha (IFN-alpha) in hairy-cell leukaemia; Hu-IFN-alpha 2 receptor expression by hairy cells and other normal and leukaemic cell types.

To investigate the possible direct effects of interferon-alpha (IFN-alpha) in hairy-cell leukaemia, IFN-alpha receptor expression by hairy cells (HCs) (11 cases) was measured by a radiolabelling technique and compared with that of MOLT-4, chronic lymphocytic leukaemia (CLL; 14 cases) and various other leukaemic and normal cell types. Purified peripheral blood (PB) and splenic HCs showed higher levels of receptor expression (approx. 1000 +/- 200 binding sites/cell; 11 cases tested) than other normal and leukaemic cells types. Purified normal PB and tonsil B cells showed low levels of receptors (approx. 120 +/- 100 binding sites/cell), while a range of B-cell leukaemias displayed intermediate levels of expression (approx. 100-500 sites/cell). In the 15 cases of CLL tested, 530 +/- 330 binding sites/cell were demonstrated, the high standard deviation reflecting the fact that approximately one third of cases had receptor levels comparable with those in HCL. Normal and HCL T cells, red cells and platelets had no demonstrable IFN receptors. It is suggested that these findings may be relevant to the efficacy of IFN in hairy-cell leukaemia.

Tolerance of one-month intranasal interferon.

Under double-blind conditions, groups of volunteers (68 in total) were allocated at random to take intranasal solutions of placebo or one of three doses of highly purified leucocyte interferon by intranasal spray twice a day for 28 days. The highest dose would have been expected to protect against experimental colds. Treatment was discontinued because of upper respiratory symptoms as often in each of the interferon groups as in the placebo group. However, it was possible to distinguish clinically between "colds" on placebo and low-dose interferon and "reactions to treatment" on high-dose interferon. The features of the reactions to treatment were a protracted build-up of local symptoms and minor epistaxis. None of the volunteers on the high-dose interferon were thought to have a definite cold, but viruses were isolated from four out of six volunteers on low-dose interferon who had definite colds. Previous experiments had also shown this dose to be insufficient to protect against experimental rhinovirus challenge. The dose of interferon that appeared to protect against virus infection caused significant unwanted effects. It is essential to find interferon preparations with less inflammatory activity before interferon can be considered for use as a long-term prophylactic against the common cold.

Measurement of interferon from in vitro stimulated lymphocytes by bioassay and monoclonal antibody-based immunoassay.

Peripheral blood mononuclear cells (PBMC) derived from healthy individuals were stimulated with u.v.-inactivated Newcastle disease virus and the cell supernatants were assayed for both antiviral activity and alpha interferon (IFN-alpha) immunoreactivity. IFN-alpha concentrations determined by two immunoradiometric assays (IRMAs) based on monoclonal antibodies that recognize different IFN-alpha subtypes correlated well together (r = 0.96) and with interferon concentrations determined by the two bioassays (r = 0.82 to 0.89), but the agreement between the results of the two bioassays was not as close (r = 0.79). As judged by the agreement between determinations on duplicate inductions of the same PBMC, the IRMAs were considerably more precise than the bioassays. Despite the use of a common IFN standard there were marked differences in the absolute titres of IFN determined by the IRMAs and bioassays, highlighting the difficulties in standardizing assays for IFN-alpha. The IRMA results suggest that there are no major differences in the spectrum of IFN-alpha subtypes produced by healthy individuals under conditions of viral stimulation.

Kinetics of internalisation and degradation of surface-bound interferon in human lymphoblastoid cells.

The binding of iodinated human interferon-alpha 2 (IFN-alpha 2) was studied on the human T cell line, Molt 4. After its initial binding to cells, the IFN is transferred to a trypsin-resistant compartment before appearing in the medium as TCA-soluble material, while the total cell-associated IFN declines to one-third of its maximum value after 3 h incubation. The Na+/H+ ionophore monensin did not prevent intracellular accumulation of IFN but did completely inhibit its breakdown. We interpret our results as evidence for receptor-mediated internalisation of IFN followed by intracellular breakdown.

Determinants recognized by murine rheumatoid factors: molecular localization using a panel of mouse myeloma variant immunoglobulins.

The structures recognized by monoclonal anti-IgG1 rheumatoid factors (RF) were localized by testing their reactivity with mutant immunoglobulins carrying gamma 1 chains that lacked either the CH1 or the CH3 domains. While optimal binding was observed in the absence of CH1, deletion of CH3 completely abolished the reactivity of all but one of the 71 monoclonal RF tested. Similar experiments were carried out with IgG2a- and IgG2b-specific RF by using variant immunoglobulins carrying various hybrid gamma 2a-gamma 2b heavy chains. It was found that both the polyclonal RF produced by autoimmune strains, MRL/MpJ-lpr and NZB/BinJ, and most of the monoclonal RF derived from normal strains, BALB/c, C57Bl/6, and 129/Sv, were directed against determinants located in a segment spanning the C-terminal 8 residues of the CH2 domain and the complete CH3 domain.

Characterization and properties of a modified human interferon-alpha containing an additional 18 amino acids at the N-terminus.

A modified human interferon-alpha 2 was produced in Escherichia coli cells infected with phage M13 mp7 containing an interferon-alpha gene. After purification by immunochromatography with the monoclonal antibody NK2, the N-terminal amino acid sequence was determined. The N-terminal methionine was absent but an additional sequence of 18 amino acids at the N-terminus was retained. The modified interferon-alpha 2 was indistinguishable from authentic interferon-alpha 2 in its ability to activate natural killer cells, to slow the growth of Daudi cells, and to confer virus resistance on heterologous cells.

An immunoradiometric assay of serum interferon using a monoclonal antibody.

An immunoradiometric assay for human interferon-alpha (HuIFN-alpha) has been adapted for the assay of low concentrations of HuIFN-alpha in human serum. The sensitivity of the assay is 5 to 10 IU/ml and the coefficient of variation less than 10%. The assay was shown to compare well with a biological (antiviral) assay in the measurement of serum interferon following intramuscular injection of HuIFN-alpha in nine volunteers. Serum interferon was also measured in the serum from 250 normal human donors. Two donors appeared to have detectable levels of HuIFN-alpha.

Purified interferon as protection against rhinovirus infection.

In a double-blind placebo-controlled study a preparation of human leucocyte interferon purified by affinity chromatography using a monoclonal antibody and applied by repeated nasal sprays reduced the incidence and severity of colds in volunteers challenged with human rhinovirus 9. Although interferon itself caused some symptoms, these were minor compared with the clinical colds. Interferon activity was still detectable in nasal washings as long as 26 hours after the last dose in about half the volunteers on active treatment.