Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides.

We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide. Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced. These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage. Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3. CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4. The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. Proteins 1999;36:217-227.

Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library.

To generate antibodies to defined cell-surface antigens, we used a large phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.

Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause.

To obtain more insight into the relationship between cyclic and regional changes in steroid receptor expression and function-related changes in the various types of cell of the normal human uterus, we performed an immunocytochemical study on paraffin-embedded sections. The distribution and intensity of immunostaining for the oestrogen receptor and the progesterone receptor in the various types of cell were semiquantitatively scored. The data were statistically compared for the different phases of the menstrual cycle and after the menopause, and for the different regions of the corpus and (endo)cervix uteri. During the menstrual cycle, significant changes in oestrogen receptor score were observed in glandular and stromal cells of endometrium basalis and functionalis and in smooth muscle cells of the myometrium. In all types of cell, oestrogen receptor expression reached a maximum in the late proliferative phase. During the early secretory phase, oestrogen receptor staining declined sharply in stromal and smooth muscle cells, whereas, in glandular epithelium, oestrogen receptor expression decreased more gradually. During mid- and late-secretory phases, an increase in oestrogen receptor staining was also observed in predecidualizing stromal cells and smooth muscle cells. Progesterone receptor numbers changed significantly in glandular epithelium but not in stromal and smooth muscle cells. Glandular progesterone receptor expression reached a maximum in the early secretory phase and was then drastically reduced. During mid- and late-secretory phases stromal cells were moderately stained for progesterone receptor in contrast to epithelial gland cells which showed no or very weak staining. No regional variations in steroid receptor distribution in endometrium and myometrium were found.(ABSTRACT TRUNCATED AT 250 WORDS)

NCI-H716 cells as a model for endocrine differentiation in colorectal cancer.

In colonic neoplasms, endocrine differentiation is encountered not only in carcinoid tumors but also in adenocarcinomas, where endocrine cells may represent a distinct line of differentiation in the tumor. The significance of endocrine differentiation in colorectal cancer is not well established, partly because of the paucity of tumor cell lines which can serve as a model for studying endocrine differentiation. In this report we describe the properties of NCI-H716 cells, a cell line derived from a poorly differentiated adenocarcinoma of the caecum, under various in vitro conditions and as xenografts in athymic mice. Phenotypical properties were immunohistochemically assessed using a panel of differentiation related antibodies, and also by Northern blot analysis and by electron microscopy. Receptors for biogenic amines and peptide hormones were analyzed by ligand binding assay. These studies show that: 1. NCI-H716 cells can be undifferentiated, or show endocrine, mucin-producing or "amphicrine" properties. 2. Endocrine differentiation of NCI-H716 cells preferentially occurs in xenografts in athymic mice, which suggests that mesenchymal elements induce endocrine differentiation. 3. NCI-H716 cells express large amounts of high affinity receptors for gastrin, serotonin and somatostatin and these substances can regulate growth. Thus, NCI-H716 cells form a suitable model for the study of endocrine differentiation in intestinal epithelium and of auto- or paracrine growth regulation in intestinal neoplasia.

Is immunohistochemical analysis of oestrogen and progesterone receptors in endometrial carcinoma superior to the radioligand binding assay?

The aim of this study was to evaluate tissue and steroid receptor heterogeneity in endometrial carcinoma specimens as a possible source of discordance between biochemically assayed receptor status and response to endocrine treatment. For this purpose the oestrogen receptor (OR) and progesterone receptor (PR) levels in specimens from 16 endometrial carcinoma patients were analysed on adjacent tissue sections using both a radiochemical and an immunohistochemical assay. With immunohistochemical receptor analysis extensive tissue and tumour cell receptor heterogeneity was observed. Many tumour samples revealed up to 75 per cent contamination with benign tissue. In the majority of cases, evaluation of immunoreactivity in normal tissue elements of the specimen could explain the apparent discordance between semiquantitative immunohistochemical receptor scoring of tumour cells and radiochemical receptor assay. Immunohistochemical analysis of OR and PR in endometrial carcinoma specimens allows a more specific determination of tumour cell receptor content and hence may yield a more accurate prediction of response to endocrine therapy than the biochemical assay.

Quantification of oestrogen receptors in breast cancer: radiochemical assay on cytosols and cryostat sections compared with semiquantitative immunocytochemical analysis.

A radiochemical oestrogen receptor assay on cytosol was correlated with a radiochemical and an immunohistochemical oestrogen receptor assay using cryostat sections from 50 breast cancer specimens. Oestrogen receptors were reliably quantitated in 6 micron cryostat sections with Scatchard analysis using radiolabelled oestradiol, and a good quantitative and qualitative relation with cytosol oestrogen receptor assay was found. Parallel sections were used for routine histological tissue verification and for direct comparison with immunohistochemistry for oestrogen receptor. Specific immunoperoxidase staining with a rat monoclonal antibody was scored by semiquantitative evaluation of the staining intensity of cancer cell nuclei. Oestrogen receptor scoring was highly reproducible when performed by the same observer. The semiquantitative immunohistochemical oestrogen receptor score correlated significantly better with the radiochemical assay on sections than with cytosol assay. Oestrogen receptor in breast cancer can be reliably assayed by semiquantitative evaluation of cryostat sections immunostained for oestrogen receptor, but only if the procedure is adequately standardised. The results underline the importance of cellular heterogeneity as a cause of variation in oestrogen receptor assay evaluation in breast cancer.

Progesterone receptor quantification with radiolabeled promegestone (R 5020) in frozen sections of endometrium and breast cancer tissue.

A technique for the determination of the progesterone receptor content at sections was developed. Series of coverglass-mounted unfixed frozen sections were incubated with [3H]R5020 only, to determine total binding, or with excess unlabeled R5020, to determine non-specific binding. Ligand binding in the tissue sections was measured by liquid scintillation counting after repeated washing of the coverslips. Elution of ligand binding proteins into the incubation buffer was quantitated with the dextran-coated charcoal method. Specific ligand binding was related to the total tissue protein content which was determined on parallel, unmounted sections. Scatchard analysis showed specific saturable and high affinity (Kd = 0.01-2 nM) section-bound and soluble binding sites in cryostat sections of calf uterus, human endometrium and breast cancer samples. Ligand specificity was studied by competition of [3H]R5020 with a 100-fold excess of various steroid receptor ligands. The competition was excellent for R5020 and progesterone, negligible for estrogens and slight for androgens and corticosteroids. These binding characteristics provide evidence that with this assay progesterone receptors are determined. Exchange experiments showed that with this method total, free as well as occupied, progesterone receptors can be measured. A highly significant linear correlation, and agreement in PR status classification between assay on cytosol and sections was obtained for a series of 21 breast cancer samples. Finally, progesterone receptor analysis using cryostat sections results in the recovery of 2-3 times more PR from the same amount of tissue as compared to the use of cytosol. These results indicate that progesterone receptors can be reliably assayed with Scatchard analysis using cryostat sections, which requires less tissue than the cytosol assay. This method, which is simple and easy to perform could be of practical importance, particularly when only small tissue samples (which also have to be analyzed morphologically or histochemically) are available and when quantitative radiochemical progesterone receptor data are required for direct comparison with (immuno-) histochemical information.

Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats.

Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a [125Iodo]-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers.