Bosco Shang Wang

Inhibition of the induction of alloreactivity with mitoxantrone

A clinically active anticancer agent, mitoxantrone (MX): 1, 4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9, 10-anthracenedione dihydrochloride, was studied for its potential inhibitory effect on alloreactivity induction. Addition of MX to mixed lymphocyte cultures (MLC) not only inhibited the proliferative response of lymphocytes to alloantigens but also prevented the generation of cytolytic T lymphocytes (CTL). MX showed a long-lasting effect in vitro and acted at the inductive rather than the effector phase of the CTL response as indicated by its failure to alter the activity of those CTL already generated in MLC. MX also inhibited CTL induction in mice. However, the precursors of CTL appeared to be spared in these animals as supported by limiting dilution analysis and also because CTL could be reactivated by exposure of splenocytes to the same or different alloantigens in MLC. The present findings demonstrate that MX is a potent immunosuppressive agent and as such might prove to be clinically useful in the treatment of autoimmune diseases or find utility in the organ transplantation field.

A proposed mechanism of action of a growth hormone-specific monoclonal antibody in the enhancement of hormonal activity

The potentiation of the biological activity of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. An anti-pGH monoclonal antibody, designated PS-7.6, was generated and its effect on pGH was evaluated in hypophysectomized (hypox) rats. As expected, administration with pGH for 5 consecutive days promoted these animals to grow. The effect was further augmented when pGH was given together with PS-7.6 antibody and the enhancing ability of the antibody lasted beyond the treatment period. The growth profile of rats receiving antibody alone did not differ from that of untreated controls, indicating that PS-7.6 antibody by itself was not a growth stimulant. The possible mechanism of action of the antibody was investigated by analyzing blood and tissue samples of animals following injection with 125I-labeled pGH either in its free form or complexed with PS-7.6 antibody. As compared to the pGH levels in animals receiving free pGH, approximately a half pGH was released into circulation from the injection sites when it was given in a complex form. Furthermore, 2-4-fold increases in pGH deposition were also found in various tissues of animals treated with pGH-antibody complexes over that of respective tissues of animals receiving free pGH. Therefore, the present findings suggest that PS-7.6 antibody is capable of augmenting the somatogenesis of pGH and the effect is, at least in part, explainable by its ability in altering the pharmacokinetics and biodistribution of pGH in animals.

Low molecular weight immunopotentiators

It has long been recognized that modulation of the immune system by various agents may have potential for the management of certain infectious and neoplastic diseases. Both natural products as well as chemically synthesized compounds have been investigated for immunotherapeutic potential. Over the years, conflicting reports on the clinical efficacy of these agents have left the early promise of immunotherapy unfulfilled. However, the manipulation of the immune system to generate a desired effect is becoming feasible as the mechanisms which regulate the immune network are better understood. Much of the early work on immunotherapy concentrated on the development of immunopotentiators, agents which enhance the hosts own immune system against cancer cells or infectious pathogens. Furthermore, with the development of subunit and/or synthetic vaccines, which are often weakly immunogenic, the importance of developing agents capable of acting as adjuvants became apparent. As a result, the utility of immunopotentiators has now extended to the area of vaccines. There are a number of reviews available on immunomodulators [see Fenichel, R. L. and Chirigos, M. A. (eds) (1984), Immune Modulation Agents and Their Mechanisms, Marcel Dekker, New York]. The purpose of this article is to provide an update on low molecular weight agents capable of potentiating the immunological network. Attention will be given to those agents which have undergone significant clinical development in the areas of cancer, infectious diseases and vaccination over the past several years. These agents will be categorized as to whether they are naturally occurring or chemically synthesized.

Relationship of chemical structures of anthraquinones with their effects on the suppression of immune responses.

A series of 37 anthraquinones were evaluated for their ability to inhibit the induction of cytolytic T-lymphocytes in a mixed lymphocyte culture system, useful as a preliminary screen for immunosuppressive agents. These compounds were also tested for their ability to prevent the production of antibody in mice. It was demonstrated that 1,4-bis [(2-aminoethyl)amino]-5, 8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD, 2) derived from mitoxantrone (MX, 1) by removing hydroxyethyl groups from both side chains was extremely active in depressing immune responses in vitro and in vivo. Four additional anthraquinones related to AEAD were also identified to share similar suppressive activity. They include a Schiff base, 1,4-dihydroxy-5,8-bis[[2-[(3-pyridinylmethylene)amino]ethyl]amino] -9,10-anthracenedione; a dimer with N-terminals methylated, 1,1-[ethylenebis (iminoethyleneimino)]-bis [5,8-dihydroxy-4-[(2-methylamino-ethyl)amino] anthraquinone tetrahydrochloride; an oxazolidine, 1,4-dihydroxy-5,8-bis [[2-(2-propyl-3-oxazolidinyl)ethyl]amino] anthraquinone; and its polymeric oxazolidine, poly [5,8-dihydroxy-1,4-anthraquinonyleneiminoethylene-3,2-oxazolidine- diyltrimethylene-2,3-oxazolidinediylethyleneimino]. These compounds may warrant further consideration as candidates for the treatment of refractory autoimmune diseases and in organ transplantation.

Induction of alloreactive immunosuppression by 1,4-bis [(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (CL 232,468)

1,4-bis[2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione (AEAD) has been investigated for its potential immunosuppressive effect on cell-mediated immune responses. Addition of the compound to mixed lymphocyte cultures (MLC) not only significantly inhibited these cells from responding to alloantigens but also prevented the induction of cytolytic T lymphocytes (CTL). A structurally related compound, mitoxantrone, was also found to be active in inhibiting CTL induction. AEAD had to be present during the first 3 days of a 5-day MLC in order to produce a significant effect and it had no effect on those CTL already generated, suggesting that it acted upon induction of CTL rather than the effector phase. Lymphocytes from mice treated with the compound were incapable of responding to alloantigens in vitro and the effect was dose- and time-dependent. Furthermore, lymphocytes from treated mice were found to inhibit CTL generation from normal mouse lymphocytes, indicating that a suppressor cell population might be induced in the spleens of animals treated with the compound. The present findings clearly demonstrate that AEAD is a compound with potent immunosuppressive activity on alloreactive immune responses.

Inhibitory effect of growth-enhancing antibody on the interaction between growth hormone and growth hormone binding protein

A monoclonal antibody (mAb), designated PS-7.6, was generated in mice by immunizing these animals with recombinant porcine growth hormone (pGH). This antibody has been previously shown to enhance the growth-promoting activity of pGH in an experimental rat model. An effort was made in this report to further characterize the immunologic properties of this antibody and its effect on the interaction between pGH and GH binding protein (GHBP). This antibody was classified as IgG1 isotype and found to be highly specific to pGH in a competition radioimmunoassay (RIA). It did not recognize several other GHs including those of bovine, chicken and human origins, nor several growth related factors including prolactin, insulin, somatostatin and growth hormone releasing factor. In Western analysis, PS-7.6 mAb identified not only the 22.5 kDa recombinant pGH, but also the native pGH in swine pituitary gland. An additional 45 kDa protein was also detected in the gland by the antibody, presumably a dimer form of pGH. The association and dissociation rate constants of the antibody as determined by biospecific interaction analysis (BIA) were 2.43 x 10(4) M-1 s-1 and 1.29 x 10(-3) s-1, respectively and its affinity (Kd) was 4.85 x 10(-8) M. Furthermore, PS-7.6 mAb prevented pGH from interacting with GHBP in RIA and BIA, indicating that the antibody epitope closely associated with the pGH binding region for GHBP. Therefore, present findings suggest that a possible mechanism of action of PS-7.6 mAb in enhancing animal growth is to prevent pGH from being bound to GHBP in circulation, thus making more pGH available to tissue receptors.

Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of 111In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

Enhancement of hormonal activity with a monoclonal antibody specific to porcine growth hormone

Abstract The potentiation of the biological effects of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. A monoclonal antibody (mAb), designated PS‐7.6, was raised against pGH and repeatedly shown to enhance the responsiveness of hypophysectomized (hypox) rats to pGH. As a result, animals receiving a combination treatment of pGH and mAb PS‐7.6 together gained significantly more weight than those receiving the same doses of pGH alone. The enhancing action of the mAb was a rapid process and its effective doses ranged from 0.1 to 2 mg/injection. The ability of the antibody to augment the hormonal activity persisted beyond the 5‐day treatment period and the differences in net weight gain between treated and control animals remained significant for 28 days. Results from treatment frequency studies further suggested that pGH when complexed with mAb PS‐7.6 required fewer injections and produced a greater efficacy than being administered alone. Therefore, present findings sugg...

Modulation of the immune response to tumors by a novel synthetic compound, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide (CL 259,763)

SummaryCL 259,763, N-[4-[(4-fluorophenyl)sulfonyl]phenyl] acetamide, is an orally active compound capable of modifying the reactivity of certain lymphoid cell populations affected by the growth of a tumor. The compound augmented the response of lymphocytes from tumor-primed animals to syngeneic tumor cells, resulting in a marked increase in tumor cell destruction. Likewise, it enhanced macrophage inhibitory effects on the growth of tumor cells in vitro. These “activated” macrophages were detectable in peritoneal exudates of treated mice 4 to 12 days after receiving a single oral dose of CL 259,763, with peak activity being demonstrable by day 7. The compound also restored the alloreactivity of lymphocytes from immunodepressed mice bearing the Lieberman plasma cell tumor, possibly by interferring with suppressor cells. Macrophages and lymphocytes from treated mice released significantly more IL-1 and IL-2-like factors in culture than did the control counterparts. Sera from treated mice also possessed more colony stimulating factor than those from normal mice. Immunoadjuvant effects were evident when the compound was administered with an inactivated L1210 leukemia vaccine and it enhanced the effectiveness of cytotoxic chemotherapy when given to mice challenged with P388 murine leukemia. These immunomodulating effects of CL 259,763 may hopefully be exploited in efforts to augment the immune response of the host to a progressively growing tumor.

Proliferation of Nb2 lymphoma cells in vitro in response to interleukin-7

An attempt was made to investigate the proliferative effect of interleukin-7 (IL-7) on a rat Nb2 T-cell lymphoma line. It was demonstrated that both human and mouse IL-7 stimulated these cells to proliferate in a dose dependent fashion in culture medium containing 10% horse serum. The maximum activities of mIL-7 and hIL-7 were observed at 100 and 1000 units/ml with their half-maximal response of 10 and 50 units/ml, respectively. In a totally serum-free culture condition, mIL-7 produced a similar cellular proliferation, whereas hIL-7 was much less effective. The effectiveness of IL-7 on Nb2 cells was completely abolished by antibody to IL-7, but not by antibody to IL-2. Therefore, Nb2 cells may serve as a simple, convenient and sensitive assay for monitoring the biological activity of IL-7 in vitro. In addition, these cells are also useful for studying the lymphopoiesis of T-cell lineage regulated by IL-7.