N-isobutyryl-His-Trp-Ala-Val-D-Ala-His-Leu-NHMe (ICI 216140) a potent in vivo antaconist analogue of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue.

The GRP receptor mediated growth response in Swiss 3T3 cells has been used to identify BN/GRP antagonists. Analysis of bombesin antagonism by substance P analogues and by truncated GRP analogues revealed that deletion of the C-terminal methionine residue was important for antagonism. Des-Met analogues showing potent antagonist activity in the in vitro 3T3 system (IC50 approximately 2nM) were synthesized. Further structural modification of these peptides led to the identification of (CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3 (ICI 216140) which reduced bombesin-stimulated rat pancreatic amylase secretion to basal levels when administered subcutaneously at 2.0 mg per kg.

Functionally important conserved amino-acids in interferon-alpha 2 identified with analogues produced from synthetic genes.

A gene was chemically synthesised and expressed in Escherichia coli to produce [Ala30,32,33]IFN-alpha 2, an analogue of human alpha 2-interferon (IFN-alpha 2) which is devoid of activity on human cells. Eight additional analogues provided single changes in IFN-alpha 2 at each of these three conserved positions. No one residue is essential for activity, but both antiviral and anti-proliferative activity are particularly sensitive to changes in the side-chain of Arg33.

Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli.

A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.

Circulating immune complexes in rats bearing chemically induced tumours. II. Characterization of sera from different stages of tumour growth.

Sera from rats bearing intraperitoneal implants of an aminoazo dye-induced hepatoma were fractionated by Sephadex G200 and DEAE-cellulose ion exchange column chromatography. Isolated fractions were examined for their capacity to bind [125I]C1q as a measure of immune complex levels, and for their ability to bind soluble tumour-specific antigen as well as to react with antigens expressed at the surface of viable hepatoma cells. Elevated levels of circulating immune complexes in unfractionated serum were directly detectable during early tumour development although, following serum fractionation, immune complexes were identified at both early and late stages of tumour growth. The present findings suggest that the detection of immune complexes in unfractionated samples of late tumour-bearer serum using a C1q-binding assay is masked by the increasing production of tumour-specific antibodies and by a shift from complement fixing to non-complement-fixing tumour-specific antibodies.

Circulating immune complexes in rats bearing chemically induced tumors. I. Sequential determination during the growth of tumours at various body sites.

Circulating immune complexes were measured in sequential monitoring studies in rats bearing chemically induced tumours at different body sites. The assay employed were based upon radioimmunoprecipitation of serum factors with 125I-C1q or with an indirect test whereby the inhibition of binding of 125I-C1q to IgG aggregates by tumour-bearer sera was measured. 125I-C1q-binding immune complexes were detected at the initial phase of tumour growth and the level of this activity returned to the normal range after tumour excision. With tumour growing at subcutaneous sites or in th peritoneal cavity, serum-borne C1q-binding material, after reaching a maximum level, decreased despite tumour progression and eventually fell into sub-normal ranges at terminal stages of growth. In contrast, following intravenous tumour cell injection, immune-complex levels increased until terminal stages of disease. The present findings indicate that the measurement of C1q-binding serum factors represents a useful method for monitoring the growth and burden of experimental animal tumours.