Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different cross-linking agents: antitumor effects, in vivo fate, and tumor cell mutants.

The A-chain of the plant toxin abrin was covalently linked to monoclonal anti-Thy 1.1 antibody (OX7) with the use of either N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane hydrochloride (2IT). The SPDP reagent generates a linkage containing a disulfide bond and an amide bond, whereas the 2IT reagent generates a linkage containing a disulfide bond and an amidinium bond. The two immunotoxins were powerfully and specifically toxic to Thy 1.1-expressing murine AKR-A lymphoma cells in vitro. Both reduced the rate of protein synthesis of the cells by 50% at a concentration of 10(-11) M. However, clonogenic assays revealed that about 1% of the AKR-A cells survived treatment with high concentrations of OX7-SPDP-abrin A, whereas only about 0.1% survived treatment with similar concentrations of OX7-2IT-abrin A. Several clones of the surviving cells were isolated. Of 11 clones of cells that had survived exposure to OX7-SPDP-abrin A, 10 were resistant to further treatment with OX7-SPDP-abrin A but had normal sensitivity to OX7-2IT-abrin A. These clones expressed moderate to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. In contrast, all 10 clones of cells that had survived exposure to OX7-2IT-abrin A were substantially or entirely resistant to both immunotoxins. They expressed low to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. The 2IT-linked immunotoxin was much more effective than the SPDP-linked immunotoxin at protecting nu/nu mice against the growth of AKR-A lymphoma cells in the peritoneal site. A single iv injection of 0.3 nmol OX7-2IT-abrin A eradicated at least 99.99% of the tumor cells, as judged from the extension in the median survival time of the animals, whereas OX7-SPDP-abrin A eradicated only about 99% of the cells. The tumors that developed in the animals that received OX7-2IT-abrin A were Thy 1.1-negative, whereas those in the recipients of OX7-SPDP-abrin A generally expressed normal levels of the Thy 1.1 antigen. The difference in antitumor activity of the immunotoxins was not due to differences in their in vivo fate, inasmuch as they were cleared from the bloodstream at an identical rate and broke down at the same rate to release free antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo.

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)tolue ne (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-iminothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.

Salivary progesterone: relation to total and non-protein-bound blood levels.

The daily amounts of salivary progesterone have been determined over the complete menstrual cycle for 9 normal women. The level of progesterone during the follicular phase was about 150 pmol/l and increased significantly to about 350 pmol/l during the luteal phase of the menstrual cycle. The amounts of salivary progesterone were significantly correlated with those in blood (P less than 0.001) in paired saliva-blood specimens taken from 96 women. Although these volunteers comprised patients with benign and malignant breast disease and normal unaffected women, the relationship between salivary and blood progesterone was similar for all groups. The concentration of non-protein-bound progesterone was determined using equilibrium dialysis. To correct for serum dilution the linear relationship between the percentage of progesterone bound and the reciprocal of serum dilution has been exploited. The values of non-protein-bound progesterone obtained were significantly and linearly correlated with levels in saliva (r = 0.75, P less than 0.001, d.f. = 34) although the amount of free progesterone in blood was about five times that found in saliva.

A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody.

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was covalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 microliter serum. The method is precise, accurate and specific and can detect 19.5 pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.

The production of high affinity monoclonal antibodies to progesterone.

Using different immunisation regimens and the antigens 6 beta-hydroxyprogesterone hemisuccinate and 11 alpha-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin, we have produced 35 monoclonal antibodies against progesterone with a wide range of specificities and affinities (Ka = 8 x 10(7)-3 x 10(10) M-1). The 10 antibodies raised against the 6 beta-antigen showed no advantage in terms of specificity when compared with those raised against the 11 alpha-antigen. The four antibodies with the best binding characteristics were all derived from fusion experiments performed after long (5 booster injections) immunisation with the 11 alpha-antigen. Although the specificities of these monoclonal antibodies were similar to those of a high affinity rabbit antiserum raised against the same antigen, a measurable improvement in sensitivity was obtained when one of these four antibodies was used for radioimmunoassay.

Human CD4- CD8- alpha beta+ T cells express a functional T cell receptor and can be activated by superantigens.

The CD4 and CD8 molecules play an important role in the stimulation of T cells and in the process of thymic education. Most mature T cells express the alpha beta TCR and either CD4 or CD8; however, there is a small population of alpha beta+ TCR T cells that lack both CD4 and CD8. Little is known of the biology of the CD4- CD8- (double-negative) alpha beta+ TCR T cells or the nature of the Ag to which they may respond. These cells not only represent a novel population of T cells but also provide useful biologic tools to study the roles that CD4 and CD8 play in T cell activation. In this study we have addressed two questions. Firstly, whether CD4- CD8- alpha beta+ TCR T cells have functionally active TCR and, secondly, whether CD4 or CD8 is required for the activation of T cells by bacterial enterotoxins. Six double-negative alpha beta+ TCR T cell clones, propagated from two healthy donors, were challenged with a panel of nine bacterial enterotoxins. The V alpha and V beta usage of their TCR was determined by polymerase chain reaction. All of the CD4-CD8- clones proliferated in response to at least one of the enterotoxins, in a V beta-specific manner. The proliferative response of the CD4-CD8- alpha beta+ TCR T cell clones was similar in magnitude to that exhibited by CD4+ T cell clones of known V beta expression. These data clearly show that the CD4 and CD8 molecules are not required for the activation of untransformed human T cells by bacterial enterotoxins. Furthermore, these results indicate that CD4-CD8- alpha beta+ TCR T cells, normally present in all individuals, are not functionally silent, because they can be stimulated via their TCR. Their physiologic role, like that of gamma delta T cells, remains to be elucidated.

Serum carcinoembryonic antigen in the diagnosis and prognosis of women with breast cancer.

Serum carcinoembryonic antigen (CEA) has been measured in 628 patients before and 577 patients after treatment for breast cancer. These came from an unselected sequence of 730 women, subsequently diagnosed as having stage I or II breast cancer, referred to Guy's Hospital over a period of nearly 5 yr. CEA was also measured in serum from 238 ostensibly healthy volunteers and 65 women with benign breast disease. CEA measurements were of no diagnostic value. There were more patients with breast cancer with values in excess of 10 ng/ml measured preoperatively (7%) or after mastectomy (5%) than in controls (3%), but the difference is of marginal significance. High levels of CEA were not consistently associated with pathological stage or histological grade. Mastectomy was not associated with any significant change in the distribution of CEA levels. Patients with stage II disease and pre-operative CEA levels over 10 ng/ml has a faster recurrence rate than those with levels of less than 2.5 ng/ml. High levels were also associated with reduced survival. However, such patients comprised about 5% of women presenting with early breast cancer, so that the use of CEA measurements for prognosis is of limited value.

Selective cytotoxic activity of immunotoxins composed of a monoclonal anti-Thy 1.1 antibody and the ribosome-inactivating proteins bryodin and momordin.

The ribosome-inactivating proteins, bryodin, from Bryonia dioica, and momordin, from Momordica charantia, were coupled by a disulphide bond to a monoclonal anti-Thy 1.1 antibody (OX7). Both immunotoxins were specifically cytotoxic to the Thy 1.1-expressing mouse lymphoma cell line AKR-A in vitro. The OX7-bryodin immunotoxins were the more powerfully toxic and reduced protein synthesis in AKR-A cells by 50% at a concentration of 1-4 x 10(-11) M as compared with 1 x 10(-9) M for the OX7-momordin immunotoxins. Neither of the immunotoxins was toxic to mouse lymphoma EL4 cells, which lack the Thy 1.1 antigen, at concentrations up to 3 x 10(-8) M. Further, bryodin and momordin immunotoxins made from an antibody (R10) of irrelevant specificity were without effect on AKR-A cells.