Highly reduced binding to high and low affinity mouse Fc gamma receptors by L234A/L235A and N297A Fc mutations engineered into mouse IgG2a.

The effects of the Fc silencing mutations such as leucine (L) to alanine (A) substitution at the position 234 and 235 (LALA) and the alanine (A) to asparagine (N) substitution at position 297 (N297A) are well investigated for human IgG. However, the effects of the same two silencing Fc mutations in a mouse IgG backbone are not yet well investigated in respect to binding to mouse Fc gamma receptors (FcγRs), complement and subsequent effector functions. By using a mouse IgG2a tool antibody directed against mouse OX40L, we demonstrate a strongly reduced binding of the two Fc mutants to high and low affinity recombinant and cell expressed mouse FcγRs, when compared to the mouse IgG2a with the wild type (wt) backbone. Reduced FcγR binding by the two investigated Fc mutants could further be confirmed on primary mouse macrophages expressing their native FcγRs. In addition, we reveal that the LALA and N297A mutations in the mIgG2a also slightly reduced binding to C1q of human origin. Thus, here we provide experimental evidence that the two investigated Fc mutations in the mouse IgG backbone lead to similar "silencing" properties as previously demonstrated for the human IgG and thus represent a useful method to alter effector functions in tool antibodies to be used in mouse models.

Synthesis of derivatives of the novel cyclophilin-binding immunosuppressant sanglifehrin A with reduced numbers of polar functions.

The syntheses and the biological activities of 53-deoxo sanglifehrin A (2) and 61-deoxy octahydrosanglifehrin A (3) are described. Compound 2 shows intracellular cyclophilin (CyP)-binding and immunosuppressive activity in the mixed-lymphocyte reaction (MLR) similar to that of sanglifehrin A (1). Compound 3 is much less active in the MLR despite unchanged intracellular CyP-binding. This indicates that the 53-keto group is not necessary for immunosuppressive activity, while the 61 hydroxy group is required.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptococcus neoformans via cyclophilin-dependent inhibition of calcineurin.

Cyclosporine (CsA) is an immunosuppressive and antimicrobial drug which, in complex with cyclophilin A, inhibits the protein phosphatase calcineurin. We recently found that Cryptococcus neoformans growth is resistant to CsA at 24 degrees C but sensitive at 37 degrees C and that calcineurin is required for growth at 37 degrees C and pathogenicity. Here CsA analogs were screened for toxicity against C. neoformans in vitro. In most cases, antifungal activity was correlated with cyclophilin A binding in vitro and inhibition of the mixed-lymphocyte reaction and interleukin 2 production in cell culture. Two unusual nonimmunosuppressive CsA derivatives, (gamma-OH) MeLeu(4)-Cs (211-810) and D-Sar (alpha-SMe)(3) Val(2)-DH-Cs (209-825), which are also toxic to C. neoformans were identified. These CsA analogs inhibit C. neoformans via fungal cyclophilin A and calcineurin homologs. Our findings identify calcineurin as a novel antifungal drug target and suggest nonimmunosuppressive CsA analogs warrant investigation as antifungal agents.

A novel anti-inflammatory drug, SDZ ASM 981, for the treatment of skin diseases: in vitro pharmacology.

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

Sanglifehrins A, B, C and D, novel cyclophilin-binding compounds isolated from Streptomyces sp. A92-308110. I. Taxonomy, fermentation, isolation and biological activity.

A novel class of macrolides for which the name sanglifehrins is proposed, has been discovered from actinomycete strains based on their high affinity binding for cyclophilin A (CypA), an immunophilin originally identified as a cytosolic protein binding cyclosporin A (CsA). The sanglifehrins were produced by Streptomyces sp. A92-308110. They were isolated and purified by extraction and several chromatographic, activity-guided steps. Sanglifehrins A and B exhibit a 10 to approximately 20 fold higher affinity for CypA than CsA, whereas the affinity of sanglifehrins C and D for CypA is comparable to that of CsA. Sanglifehrins exhibit a lower immunosuppressive activity than CsA when tested in the mixed lymphocyte reaction. Their in vitro activity indicates that they belong to a novel class of immunosuppressants.

Macrolide Analogues of the Novel Immunosuppressant Sanglifehrin: New Application of the Ring-Closing Metathesis Reaction.

Macrocycles containing a conjugated 1,3-diene moiety have been synthesized for the first time in good yields by the ring-closing metathesis reaction [Eq. (1)]. The new compounds represent cyclophilin-binding, simplified analogues of the macrocyclic core of sanglifehrin A, an immunosuppressant which binds with high affinity to cyclophilin.

Preparation and immunosuppressive activity of 32-(O)-acylated and 32-(O)-thioacylated analogues of ascomycin.

A series of 32-(O)-acylated and 32-(O)-thioacylated derivatives of the antibiotic ascomycin (1) have been synthesized. These readily accessible analogues exhibit potent immunosuppressive activity in vitro, as measured by an interleukin-2 reporter gene assay and the mixed lymphocyte reaction. Such molecules are expected to have a therapeutic potential in chronic inflammatory diseases of the airways such as asthma.

Contribution of donor-specific antibodies to acute allograft rejection: evidence from B cell-deficient mice.

BACKGROUND: The role of T lymphocytes in acute allograft rejection is well established. The involvement of B lymphocytes in this process, however, is more controversial. A series of reports showed that mice without a functional B-cell compartment rejected allografts with the same kinetics as control animals. In rats, however, alloantibodies were found to play a decisive role in allograft rejection. To provide an explanation for the discrepant results, we readdressed the role of B cells and antibodies in mice with disrupted immunoglobulin mu chain genes. The use of cyclosporine (CsA), which strongly suppresses T cells, allowed us to focus specifically on the function of B cells. METHODS: C57BL/6 mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (referred to as microMT/microMT mice) and microMT/+ control mice with one functional mu chain were heterotopically transplanted with fully MHC-disparate BALB/c hearts. CsA was administered subcutaneously by Alzet osmotic pumps. Normal and immune serum specific for donor hearts was given to assess the role of antibodies in the rejection process. RESULTS: Both B cell-deficient microMT/microMT and heterozygous microMT/+ mice were found to reject transplanted hearts within a similar period of time. In contrast, when T cells were partially suppressed with CsA, graft survival was significantly prolonged in microMT/microMT mice as compared with heterozygous controls. Passive transfer of donor-specific immune serum, obtained from microMT/+ animals rejecting allogeneic hearts, to CsA-treated microMT/microMT mice significantly accelerated allograft rejection as opposed to recipients treated with normal serum. CONCLUSIONS: B lymphocytes and antibodies play an important role in acute allograft rejection particularly when the dominant T-cell compartment is partially suppressed.

SDZ RAD, a new rapamycin derivative: synergism with cyclosporine.

BACKGROUND: SDZ RAD is a new rapamycin analog with potent immunosuppressive activity. Compounds of the rapamycin class differ in their mode of action from cyclosporine, thus providing a rationale for potential synergism of these two potent immunosuppressants. METHODS: The two-way mouse mixed lymphocyte reaction (BALB/c-CBA strain combination) was applied. Orthotopic kidney and heterotopic heart allografting was performed in the stringent DA-to-Lewis rat strain combination, with administration of compounds orally as microemulsion preconcentrate (i.e., Neoral in the case of cyclosporine). RESULTS: Isobologram analysis of checkerboard titrations of SDZ RAD and cyclosporine in two-way mouse mixed lymphocyte reactions indicates a synergistic interaction in vitro. In vivo, the minimal effective dose of microemulsion cyclosporine giving long-term graft survival was 5.0 mg/kg/day; for SDZ RAD, the minimal effective dose was 5.0 mg/kg/day in kidney transplantation and >5.0 mg/kg/day in heart transplantation. Long-term allograft survival was noted for combinations of microemulsion cyclosporine administered at 1.0 or 2.0 mg/kg/day and SDZ RAD given at between 0.5 and 2.0 mg/kg/day. The index of synergy in different combinations ranged between 0.3 and 0.7. CONCLUSIONS: SDZ RAD and cyclosporine show synergism in immunosuppression, both in vitro and in vitro. They form a promising synergistic drug combination in allotransplantation.

SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo.

BACKGROUND: This report describes the preclinical pharmacological profile of the new rapamycin analog, SDZ RAD, i.e., 40-O-(2-hydroxyethyl)-rapamycin. METHODS: The pharmacological effects of SDZ RAD were assessed in a variety of in vitro and in vivo models, which included an autoimmune disease model as well as kidney and heart allotransplantation models using different rat strain combinations. RESULTS: SDZ RAD has a mode of action that is different from that of cyclosporine or FK506. In contrast to the latter, SDZ RAD inhibits growth factor-driven cell proliferation in general, as demonstrated for the in vitro cell proliferation of a lymphoid cell line and of vascular smooth muscle cells. SDZ RAD is immunosuppressive in vitro as demonstrated by the inhibition of mouse and human mixed lymphocyte reactions and the inhibition of antigen-driven proliferation of human T-cell clones. The concentrations needed to achieve 50% inhibition in all of these assays fall into the subnanomolar range. SDZ RAD is effective in the in vivo models when given by the oral route in doses ranging between 1 mg/kg/day and 5 mg/kg/day. When compared with rapamycin, the in vitro activity of SDZ RAD is generally about two to three times lower; however, when administered orally, SDZ RAD is at least as active in vivo as rapamycin. CONCLUSIONS: In conclusion, SDZ RAD is a new, orally active rapamycin-derivative that is immunosuppressive and that efficiently prevents graft rejection in rat models of allotransplantation. SDZ RAD has therefore been selected for development for use in combination with cyclosporine A to prevent acute and chronic rejection after solid organ allotransplantation.

Cymbimicin A and B, two novel cyclophilin-binding structures isolated from actinomycetes.

Two novel metabolites, cymbimicins A and B, were isolated from the culture broth of a strain of Micromonospora sp. by screening for cyclophilin binding metabolites from actinomycete strains. Cymbimicin A binds to cyclophilin A with a high affinity six fold lower than to that of cyclosporin A. The binding affinity of cymbimicin B is about 100 times lower. The taxonomy of the producing strain, fermentation, isolation, physical and biological properties and structure elucidation are described.

Anti-lymphoproliferative activity of brown adipose tissue of hibernating ground squirrels is mainly caused by AMP.

A fraction with mol wt < 1 kDa was obtained from the brown fat of hibernating ground squirrels (Citellus undulatus) by means of delipidization, acid extraction, ultracentrifugation and ultrafiltration. This fraction suppressed the proliferation of mouse lymph node cells under standard mitogenic stimuli for T lymphocytes. In contrast, the fraction with mol wt < 1 kDa obtained from the brown fat of active ground squirrels in spring did not display such activity. Further HPLC purification of the biologically active fraction and chemical and structural analysis of its most potent antilymphoproliferative component revealed that this is adenosine 5'-monophosphate (AMP). These data lend support to the notion that in hibernating mammals AMP originating, at least partly, from the brown fat down-regulates the seasonally-dependent proliferation of the thymus.

Nephrotoxicity of cyclosporin A and FK506: inhibition of calcineurin phosphatase.

Cyclosporin A (CsA; 50 mg/kg) and Fujimycine (FK506; 5 mg/kg), but not the related macrolide immunosuppressant rapamycin (5 mg/kg), caused a reduction of glomerular filtration rate, degenerative changes of proximal tubular epithelium, and hypertrophy of the juxtaglomerular apparatus in male Wistar rats when given for 10 days. The molecular mechanisms of CsA and FK506 toxicity were investigated. Cyclophilin A and FK506-binding protein, the main intracytoplasmic receptors for CsA and FK506, respectively, were each detected in renal tissue extract. In the kidney, high levels of immunoreactive and enzymatically active calcineurin were found which were inhibited by the immunosuppressants CsA and FK506, but not by rapamycin. Finally, specific immunophilin-drug-calcineurin complexes formed only in the presence of CsA and FK506, but not rapamycin. These results suggest that the nephrotoxic effects of CsA and FK506 is likely mediated through binding to renal immunophilin and inhibiting calcineurin phosphatase.

Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus (HIV) type 1: interference with HIV protein-cyclophilin A interactions.

Cyclosporins, in particular the nonimmunosuppressive derivative SDZ NIM 811, exhibit potent anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. SDZ NIM 811 interferes at two stages of the viral replication cycle: (i) translocation of the preintegration complex to the nucleus and (ii) production of infectious virus particles. Immunosuppressive activity is not correlated with anti-HIV-1 activity of cyclosporins. However, binding to cyclophilin A, the major cellular receptor protein of cyclosporins, is a prerequisite for HIV inhibition: all structural changes of the cyclosporin A molecule leading to loss of affinity to cyclophilin abolished the antiviral effect. Cyclosporin derivatives did not interact directly with HIV-1 proteins; cyclophilin was the only detectable receptor protein for antivirally active cyclosporins. There is no evidence that inhibition of HIV occurs via a gain of function of cyclophilin in the presence of cyclosporins: the complex of cyclophilin A with SDZ NIM 811 does not bind to calcineurin or to any other viral or cellular proteins under conditions in which calcineurin binding to the cyclophilin A-cyclosporin A complex is easily detectable. Thus, the loss of function caused by binding of cyclosporins to cyclophilin seems to be sufficient for the anti-HIV effect. Cyclophilin A was demonstrated to bind to HIV-1 p24gag, and the formation of complexes was blocked by cyclosporins with 50% inhibitory concentrations of about 0.7 microM. HIV-2 and simian immunodeficiency virus are only weakly or not at all inhibited by cyclosporins. For gag-encoded proteins derived from HIV-1, HIV-2, or simian immunodeficiency virus particles, cyclophilin-binding capacity correlated with sensitivity of the viruses to inhibition by cyclosporins. Cyclophilin A also binds to HIV-1 proteins other than gag-encoded proteins, namely, p17gag, Nef, Vif, and gp120env; the biological significance of these interactions is questionable. We conclude that HIV-1 Gag-cyclophilin A interaction may be essential in HIV-1 replication, and interference with this interaction may be the molecular basis for the antiviral activity of cyclosporins.

Molecular mechanisms of immunosuppression by cyclosporins.

Despite the successful clinical application of the immunosuppressive drug cyclosporin A (CsA, Sandimmun), its precise mechanism of action in the process of T cell activation remains elusive. CsA binds to the high-affinity cytosolic receptor cyclophilin whose peptidyl-prolyl cis-trans isomerase activity is inhibited upon binding. The linkage of this effect with the inhibition of the T cell receptor-mediated signal transduction pathway, which leads to a suppression of lymphokine gene transcription, is still unclear. We analyzed the relationship between cyclophilin-binding and immunosuppressive activity (e.g., effect on IL-2 transcription) of cyclosporin derivatives in vitro. The results show that binding to cyclophilin is required, but not sufficient for immunosuppression. Cyclosporin analogues which completely lack immunosuppressive activity but fully retained their cyclophilin-binding capacity antagonize the immunosuppressive activity of CsA. These derivatives inhibit the isomerase activity of cyclophilin, which clearly demonstrates that inhibition of the cyclophilin isomerase activity does not lead to immunosuppression. In analogy to the other immunosuppressants of microbial origin, FK-506 and rapamycin, a specific structure of the "effector" domain of CsA, which is unrelated to the cyclophilin-binding domain, determines the biological activity. In the nucleus, CsA interferes with the DNA-binding of inducible transcription factors to their respective DNA motifs within lymphokine promoters by affecting intracellular translocation of transcription factor subunits.

Phage display as a rapid gene expression system: production of bioactive cytokine-phage and generation of neutralizing monoclonal antibodies.

Proteins, such as hormones, enzymes, or antibody binding sites, can be expressed in an active conformation on the surface of filamentous bacteriophage. Although the phage display technology was originally developed for binding studies, we demonstrate here that this technique can rapidly provide cytokines for studies of biological activity and for raising neutralizing monoclonal antibodies. A phage M13-based cloning vector was constructed that facilitated the expression of human interleukin 3 (hIL-3) on the phage surface. The recombinant phage could stimulate the growth of the hIL-3 dependent cell line M-07, providing evidence for the display of hIL-3 in an active form. Injection of recombinant phage into mice provoked an immune response to hIL-3, and neutralizing monoclonal antibodies directed against native hIL-3 could be established from these mice with a high frequency.

Autocrine or paracrine transforming growth factor-beta modulates the phenotype of chick embryo sternal chondrocytes in serum-free agarose culture.

Sternal chondrocytes of 17-day-old chick embryos in serum-free agarose culture secrete transforming growth factor-beta. Media conditioned by such cells prevent serum-induced chondrocyte hypertrophy and cause a phenotypic modulation in serum-free culture which is similar to that observed for chondrocytes in monolayer culture. The modulated cells lose the round shape of differentiated chondrocytes and increasingly with time resemble tendon fibroblasts embedded into agarose. In addition, they produce less matrix macromolecules which include collagen I rather than cartilage collagens II, IX, X, and XI. All of these effects are abolished upon addition to the conditioned media of a monoclonal antibody against recombinant human transforming growth factor-beta 2. The same factor caused effects closely similar to those elicited by conditioned media. Therefore, the phenotypic modulation in adhesion-dependent cultures of chondrocytes in vitro does not directly result from cell-matrix interactions but can be produced also in suspension culture under the direction of appropriate diffusible stimuli that include transforming growth factor-beta. In addition, the results support the concept of transforming growth factor-beta as a multifunctional cytokine acting differently on cells of the same developmental origin depending on their stage of differentiation.

Evidence for shared receptor proteins for human interleukin-3 and granulocyte-macrophage colony-stimulating factor in the human M-07 cell line.

The biologic response of the human leukemia cell line M-07 to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 4 (IL-4) is mediated by a low number of high affinity receptors. Cross-competition studies revealed that IL-3 and GM-CSF partially inhibited the specific binding of the heterologous radiolabeled ligand, whereas IL-4 binding was not affected by these cytokines. The molecular mechanism of cross-competition was investigated by chemical crosslinking and immunoprecipitation. Trimolecular receptor complexes consisting of a major 73kDa and two minor 120 and 128kDa membrane proteins for IL-3, and a major 84kDa and two minor 120 and 130 kDa proteins for GM-CSF were found on M-07 cells. The 73 and 84kDa proteins represent distinct and non-linked membrane proteins and are identical with the cloned, low affinity IL-3 and GM-CSF receptor proteins (Gearing et al, 1989, Hayashida et al, 1990). The higher molecular weight proteins share common binding sites as evidenced by immunoprecipitation of double-crosslinked membranes. The 120/128kDa proteins are most likely identical with the recently cloned and shared beta-subunit of the IL-3 and GM-CSF receptor (Kitamura et al, 1991) containing a single or two IL-3 and/or GM-CSF molecules.