Anti-cyclosporine monoclonal antibodies and their anti-idiotopic counterpart: structure and biological activity.

In order to study the structural and functional mimicry of an antigen by anti-idiotypic antibodies, we generated anti-idiotopic monoclonal antibodies (anti-Id mAbs) against a mAb (R45-45-11) with specificity for the immunosuppressive cyclic undecapeptide cyclosporine (Cs; Sandimmune). Three out of five anti-Id mAbs inhibited the binding of Cs to the anti-Cs mAb R45-45-11. All anti-Id mAbs cross-reacted only with one (anti-Cs mAb V45-271-10) out of 19 anti-Cs mAbs. The anti-Cs mAb V45-271-10 recognizes an epitope on the Cs molecule which is very similar to that recognized by R45-45-11. R45-45-11 and V45-271-10 differ only by one amino acid in the variable region. The anti-Id mAbs which recognize combining site-associated idiotopes (Ids) reverse the blocking effect of the anti-Cs mAb R45-45-11 on Cs immunosuppression in vitro. The sequences of the variable regions of heavy and light chain of one anti-Id mAb were determined. X-ray analysis of the corresponding Fab fragment, either alone or complexed with the Fab fragment of the Id, is currently in progress.

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.

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.

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.

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.

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.

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.

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.

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.

Characterization of a major human antibody clonotype (1A) by monoclonal antibodies to combining site-associated idiotopes.

To identify major human antibody clonotypes with specificity to N-acetyl-D-glucosamine (GlcNAc), affinity-purified antibody preparations of different human individuals were analyzed by isoelectric focusing (IEF). A major clonotype (1A) was identified representing 7% of all anti-GlcNAc antibodies of one donor (no. 371). Anti-GlcNAc antibodies of donor 371 were used as immunogen to prepare monoclonal anti-idiotopic antibodies (mAb). Two anti-idiotopic mAb were specific for the major clonotype 1A, as shown by blotting of the anti-GlcNAc antibodies after IEF in thin-layer agarose to nitrocellulose filters and staining with either iodinated antigen or iodinated anti-idiotopic mAb. The two anti-idiotopic mAb 13F15 and 10F59 were further analyzed with respect to the antigenic determinant which they recognize. Both were directed to combining site-related determinants as shown by inhibition studies. Although 13F15 has a 500-fold higher binding capacity (relative affinity) equal amounts do not inhibit more than 50% of 10F59 binding, suggesting that the two mAb detect two closely related, but not identical, idiotopes in the antigen combining site of clone 1A. Although clonotype 1A is a unique antibody exclusively found in IEF of donor 371, the idiotope 1A.1, defined by mAb 13F15, is a recurrent determinant detectable on different anti-GlcNAc spectrotypes in various human sera.

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells.

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.

Isotype restriction of idiotopes associated with human anti-streptococcal A carbohydrate antibodies.

Sharing of idiotopes between IgG and IgM antibodies has been described with antibodies of various specificities in the literature. However, this does not seem to be the rule for human IgG and IgM antibodies with specificity to streptococcal A carbohydrate. Six idiotopes were described which are associated either with IgM or with IgG antibodies to one of the two major epitopes of streptococcal A carbohydrate: N-acetyl-D-glucosamine or alpha(1,2), alpha(1,3)-linked rhamnose oligomers. Some of the idiotopes are widely cross-reactive with antibodies of the corresponding specificity in other individuals. They were constantly found to be restricted to either IgM or IgG in all individuals tested so far. We discuss several alternatives to explain this finding and conclude that switching from IgM to IgG is either a very rare event in human B cells of this specificity, or leads to loss and gain of idiotopes and/or specificity due to frequent somatic mutations. The phenomenon of idiotope restriction to certain isotypes might be important if anti-idiotopic monoclonal antibodies are considered for use in therapeutical approaches aimed at the manipulation of antibody production.

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.

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.

Characterization of minor and major idiotopes associated with human antibodies to N-acetyl-D-glucosamine.

Human antibodies to N-acetyl-D-glucosamine (GlcNAc) were analysed by conventional and monoclonal antiidiotypic antibodies. These were prepared to the antibody secreted by the human lymphoblastoid cell line B17 and to the antibody isolated from an individual serum containing a prominent clonotype 1A, both with specificity for GlcNAc. Idiotypes and idiotopes associated with these antibodies have the following properties: 1) their frequency of expression in the human population varies between rare (approximately 1.5%) and frequent (greater than 80%); 2) they have a high degree of association with antibody specificity for GlcNAc (50-100%); 3) each idiotope appears to be expressed independently of others; 4) one idiotope may be associated with a variety of clonotypes; and 5) idiotopes can be identified which are both frequently expressed in the population and associated with major proportions of the antibodies in single individuals. These latter idiotopes may be useful for studies on the modulation of human immune responses by antiidiotypic antibodies.

Developmental regulation of granulocyte-macrophage colony-stimulating factor production during human monocyte-to-macrophage maturation.

Cells of the macrophage lineage are a major source of various cytokines and hematopoietic growth factors. With regard to the growth factors acting on cells of their own lineage, macrophage colony-stimulating factor (M-CSF) has been proven to be secreted by monocytes (MO) and macrophages (MAC), whereas the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by human MO/MAC is under debate. Here we report that in elutriation-purified MO, as well as in MAC derived from cultured MO, GM-CSF m-RNA was regularly induced by LPS. In MO the GM-CSF message was still detectable 18 h after stimulation under serum-free conditions, but in contrast was already lost at this time point in MAC. Secreted GM-CSF protein was detected in the culture medium using a sandwich ELISA. Furthermore, a factor-dependent cell line (M-07) was used for a biological assay. Here, a neutralizing anti GM-CSF antibody specifically blocked the proliferation-inducing activity of MO/MAC supernatants. Whereas only small amounts of GM-CSF were detected in MO, its secretion increased severalfold upon MO-to-MAC differentiation in vitro. A similar increase upon in vitro maturation of MO was observed for the production of granulocyte colony-stimulating factor. The highest amounts of GM-CSF (up to 2.8 ng/10(6) cells) were produced by MAC that had been derived from MO cultured under serum-free conditions in the presence of 0.5 mg/ml albumin as the only medium supplement.