Biomedical subjects
Publications and source records attributed to H Waldmann.
A Wieland-Miescher type ketone and a tetramethoxyaryl derivative are the key building blocks for the enantioselective total synthesis of nakijiquinone C (1). The nakijiquinones are the only natural products known that selectively inhibit the Her-2/Neu tyrosine kinase, a protooncogene product that is vastly overexpressed in about 30 % of primary breast, ovary, and gastric carcinomas.
BACKGROUND: The potential therapeutic benefits of CD3 monoclonal antibodies, such as OKT3, have been limited by their immunogenicity and their propensity to activate a severe cytokine release syndrome. This has constrained the clinical use of OKT3 to the treatment of acute rejection episodes of organ allografts. METHODS: We have humanized a rat CD3 antibody and created a single amino acid substitution in position 297 of the IgG1 heavy chain to prevent glycosylation and, consequently, binding of the therapeutic antibody to Fc receptors and to complement. This antibody has been given as first line antirejection therapy in nine kidney transplant recipients with biopsy-proven acute rejection episodes. RESULTS: None of the patients demonstrated any antiglobulin response nor any significant cytokine release syndrome. Seven of the nine showed evidence of resolution of their rejection, although some patients experienced re-rejection. CONCLUSIONS: These findings suggest that CD3 antibodies can be engineered to lose their toxicity while retaining their potency as immunosuppressants. Nonactivating humanized CD3 monoclonal antibodies now merit further investigation in the management of transplant patients and in therapy of autoimmune diseases.
BACKGROUND: Campath 1H is a depleting, humanized anti-CD52 monoclonal antibody that has now been used in 31 renal allograft recipients. The results have been very encouraging and are presented herein. METHODS: Campath 1H was administered, intravenously, in a dose of 20 mg, on day 0 and day 1 after renal transplant. Low-dose cyclosporine (Neoral) was then initiated at 72 hr after transplant. These patients were maintained on low-dose monotherapy with cyclosporine. RESULTS: At present, the mean follow-up is 21 months (range: 15-28 months). All but one patient are alive and 29 have intact functioning grafts. There have been six separate episodes of steroid-responsive rejection. One patient has had a recurrence of her original disease. Two patients have suffered from opportunistic infections, which responded to therapy. One patient has died secondary to ischemic cardiac failure. CONCLUSIONS: Campath 1H has resulted in acceptable outcomes in this group of renal allograft recipients. This novel therapy is of equal efficacy compared to conventional triple therapy, but allows the patient to be steroid-free and to be maintained on very-low-dose immunosuppressive monotherapy.
BACKGROUND: Antiglobulin responses are a significant limitation to the repeated use of murine monoclonal antibodies for treatment of transplant rejection. It is hoped that these might be largely overcome by using antibodies genetically engineered to resemble human antibodies. METHODS: We have compared the responses in patients treated with the CD52 monoclonal antibodies CAMPATH-1G (rat IgG2b) or its humanized derivative, CAMPATH-1H (human immunoglobulin G1). RESULTS: A majority of patients (15 of 17) made responses to the rat antibody, but there were no detectable responses to the humanized antibody (0 of 12). CONCLUSIONS: Although anti-idiotype responses are theoretically possible against humanized therapeutic antibodies and are especially likely to be provoked by cell-binding antibodies, these data show that humanization offers a significant reduction in immunogenicity, potentially allowing repeat courses of treatment.
BACKGROUND: Multiple sclerosis results from T-cell-dependent inflammatory demyelination of the central nervous system. Our objective was long-term suppression of inflammation with short-term monoclonal antibody treatment. METHODS: We depleted 95% of circulating lymphocytes in 27 patients with multiple sclerosis by means of a 5-day pulse of the humanised anti-CD52 monoclonal antibody, Campath-1H. Clinical and haematological consequences of T-cell depletion, and in-vitro responses of patients' peripheral-blood mononuclear cells were analysed serially for 18 months after treatment. FINDINGS: Radiological and clinical markers of disease activity were significantly decreased for at least 18 months after treatment. However, a third of patients developed antibodies against the thyrotropin receptor and carbimazole-responsive autoimmune hyperthyroidism. The depleted peripheral lymphocyte pool was reconstituted with cells that had decreased mitogen-induced proliferation and interferon gamma secretion in vitro. INTERPRETATION: Campath-1H causes the immune response to change from the Th1 phenotype, suppressing multiple sclerosis disease activity, but permitting the generation of antibody-mediated thyroid autoimmunity.
The CD40-CD40 ligand (CD40L) interaction is a key event in the initiation of an adaptive immune response, and as such the therapeutic value of CD40L blockade has been studied in many experimental models of tissue transplantation and autoimmune disease. In rodents, transplantation of allogeneic tissues under the cover of anti-CD40L Abs has resulted in prolonged graft survival but not tolerance. In this report, we show that failure to induce tolerance probably results from the inability of anti-CD40L Abs to prevent graft rejection elicited by the CD8+ T cell subset. When the CD8+ T cell population is controlled independently, using anti-CD8 Abs, then tolerance is possible. Transplantation tolerance induced by anti-CD4 mAbs can often be associated with dominant regulation, manifested as infectious tolerance and linked suppression, both of which are mediated by CD4+ T cells. We show here that CD4+ T cells rendered tolerant using anti-CD40L therapy exhibit the same regulatory property of linked suppression, as demonstrated by their ability to accept grafts expressing third party Ags only if they are expressed in conjunction with the tolerated Ags. This observation of linked suppression reveals a hitherto undocumented consequence of CD40L blockade that suggests the tolerant state is maintained by a dominant regulatory mechanism. Our results suggest that, although anti-CD40L Abs are attractive clinical immunotherapeutic agents, additional therapies to control aggressive CD8+ T cell responses may be required.
Potent activators of protein kinase C in fibroblasts: This property was determined for several indolactam V analogues (1) with a new cell-based assay system. This tumor-promoting indole alkaloid and analogues thereof can be synthesized efficiently on the solid phase. The key steps of the combinatorial approach are a regioselective amination of the indole ring and an enantioselective enzymatic reaction.
Disparity for HLA in unrelated donor bone marrow transplantation (BMT) increases the risk of graft rejection and graft-versus-host disease (GVHD) and may compromise transplant outcome. We have compared the outcome of matched and mismatched transplants from unrelated donors in 137 children with acute lymphoblastic leukemia (ALL). Their disease status was complete remission (CR)-1, 24 patients; CR-2, 88 patients; CR-3, 18 patients; CR-4, 2 patients; and relapse, 5 patients. CAMPATH monoclonal antibodies were used for T-cell depletion and cyclosporin A was given to 134 children together with short-course methotrexate in 43, mainly when there was HLA disparity. Fifty-two donor/recipient pairs were HLA-mismatched, 41 at HLA-A and -B and 11 at HLA-DR and -DQ loci. Overall graft failure was increased in recipients of marrow mismatched at either HLA-A, -B, -DR, or -DQ (15.7% v 4.8%; P =.057) mainly because there was a higher proportion of children with primary graft failure (11. 8% v 1.2%; P =.012). The presence of an HLA-C locus mismatch did not independently increase the likelihood of graft failure. There was no significant difference in the incidence of acute GVHD >/= grade 2 between the matched and mismatched groups (P =.849). For patients in CR-2, the risk of relapse post-BMT was significantly lower if leukemic relapse occurred off-treatment (P =.005). The Kaplan-Meier overall and leukemia-free survival (LFS) estimates for recipients of matched and mismatched BMT, respectively, at 36 months were 49% versus 42% (P =.380) and 45% versus 40% (P =.654). Although HLA mismatching results in an increased occurrence of primary graft failure with T-cell-depleted allografts, it allows more donors to be identified rapidly for children with ALL without compromising overall transplant outcome.
Human CD52 (CAMPATH-1 antigen) is an abundant surface molecule on lymphocytes and a favoured target for lymphoma therapy and immunosuppression. It comprises a small glycosylphosphatidylinositol (GPI) anchored peptide to which a large carbohydrate moiety is attached. Structurally similar proteins include the proposed mouse homologue, B7 antigen (B7-Ag; not to be confused with the CD28 ligand), and human and mouse CD24. Sequence similarities between CD52 and B7-Ag precursors are concentrated over the signal peptides and the sequences cleaved during GPI attachment. While the short mature peptides are not apparently homologous, the N-linked glycosylation site is retained in both. We describe similarities in exon-intron organisation, syntenic chromosome positions (human CD52, 1p36; mouse B7-Ag, chromosome 4, between Dsil and D4Nds16) and sequence homology in the promoter regions which strongly suggests that B7-Ag is the mouse homologue of CD52. The structure of these genes is also similar to that of mouse CD24, suggesting a common ancestor. Promoter activities and transcription start sites were also analysed. These results suggest that human CD52 and mouse B7-Ag gene expressions are controlled by TATA-less promoters.
CAMPATH-1 antibodies have a long and successful history in the treatment of leukaemia, autoimmune disease and transplant rejection. The first antibody to undergo "humanisation", CAMPATH-1H, permits treatment with limited patient anti-globulin response. It recognises the CD52 antigen which is a small glycosylphosphatidylinositol(GPI)-anchored protein expressed on lymphocytes and mediates cell depletion. We present the 1.9 A structure of the CAMPATH-1H Fab complexed [corrected] with an analogue of the antigenic determinant of CD52. Analysis of the CAMPATH-1H binding site reveals that in contrast to most antibodies CDR L3 plays a dominant role in antigen binding. Furthermore CDR H3, which is essential for effective antigen recognition in most antibodies, participates in only two main-chain interactions in CAMPATH-1H. The CAMPATH-1H binding site is highly basic; ionic interaction with the enthanolamine phosphate of the CD52 GPI anchor has long been hypothesised to be important in antigen binding. The structure reveals a number of important specific ionic interactions, including Lys53H but not Lys52bH as had previously been suggested. Prolonged treatment with CAMPATH-1H can lead to patient anti-idiotype responses which may be exacerbated by the unusually high number of basic residues in the antibody. This suggests that a strategy where redundant basic residues are replaced with neutral counterparts may be effective in further reducing the immunogenicity of this versatile and widely used antibody.
BACKGROUND: B-cell lymphoproliferative disorders (BLPD*) caused by Epstein-Barr virus (EBV) occurring after allogeneic bone marrow transplantation (BMT) are usually of donor origin. Treatment such as discontinuation of immunosuppression may be successful in some cases, but infusion of donor T cells results in successful eradication of EBV BLPD in most cases. METHODS AND RESULTS: We report a case of EBV positive aggressive BLPD after HLA matched sibling BMT for aplastic anaemia. The tumour completely regressed after withdrawal of cyclosporin and donor lymphocyte infusion. However, although the tumor was of donor origin, the donor serum was negative for antibodies to EBV antigens and no EBV-specific cytotoxicity was detected in donor peripheral blood mononuclear cells. The recipient was seropositive for EBV before BMT. CONCLUSIONS: We speculate that a 'second primary' EBV infection occurred involving donor cells in the recipient during BMT immunosuppression, with subsequent outgrowth of donor-derived BLPD. EBV infection may have been by an endogenous EBV isolate, from external sources, or from third party transfusions.
IL-12 is a heterodimer of two subunits, p35 and p40, encoded by separate genes that are regulated independently. To investigate the mechanisms underlying the regulation of the p35 gene, we characterized murine p35 expression in the B cell lymphoma line A20 and in bone marrow-derived dendritic cells. Multiple transcription start sites were identified in both cell types, resulting in four p35 mRNA isoforms (types I-IV) that differ in the number and position of upstream ATGs in their 5' untranslated regions. In nonstimulated cells, the predominant forms of p35 message (types II and IV) contained an additional upstream ATG, whose presence was shown to inhibit the downstream translation of the p35 subunit. After LPS stimulation, however, transcription initiated from alternate positions, so that the proportion of transcripts not containing this upstream ATG (types I and III) was significantly increased in the population of p35 mRNA. These type I and type III transcripts readily supported translation of the p35 subunit and its incorporation into bioactive IL-12. Furthermore, p35 mRNA levels were substantially up-regulated after LPS stimulation in both cell types. Thus, our results show that p35 gene expression is highly regulated by both transcriptional and translational mechanisms.
The immunogenicity of therapeutic Abs limits their long-term use. The processes of complementarity-determining region grafting, resurfacing, and hyperchimerization diminish mAb immunogenicity by reducing the number of foreign residues. However, this does not prevent anti-idiotypic and anti-allotypic responses following repeated administration of cell-binding Abs. Classical studies have demonstrated that monomeric human IgG is profoundly tolerogenic in a number of species. If cell-binding Abs could be converted into monomeric non-cell-binding tolerogens, then it should be possible to pretolerize patients to the therapeutic cell-binding form. We demonstrate that non-cell-binding minimal mutants of the anti-CD52 Ab CAMPATH-1H lose immunogenicity and can tolerize to the "wild-type" Ab in CD52-expressing transgenic mice. This finding could have utility in the long-term administration of therapeutic proteins to humans.
BACKGROUND: Preventing graft-versus-host disease (GVHD) by depletion of T lymphocytes from the stem cell graft for transplantation remains controversial, mainly because of the perceived increase in disease recurrence. METHODS: We retrospectively analyzed the outcome of 50 consecutive individuals in remission of acute lymphoblastic leukemia (n=13; 8 in complete remission [CR]1) or acute myeloblastic leukemia (n=37; 33 in CR1), who had received marrow grafts from HLA-identical siblings. The conditioning regimen included six 2-Gy fractions of total body irradiation, succeeded by cyclophosphamide at 120 mg/kg (with mesna) followed by four fractions of 1.5 Gy to lymphoid areas. Bone marrow (n=38) or peripheral blood mobilized donor mononuclear cells (n=12) were exposed ex vivo to CAMPATH-1 (IgM and complement, or IgG; antiCD52) antibodies, without any further posttransplantation immunosuppression. RESULTS: Median patient age was 31 (range 14-51) years; 12 patients were 40 or older. Thirty-two patients were male. One patient died of pulmonary hemorrhage on day 10; another died on day 29 of interstitial pneumonitis. Except for one early death, all patients engrafted. Ten (21%) of the remaining 48 who were at risk, developed GVHD. In none was it greater than grade II. Eight patients developed serious viral infections. Four died of cytomegalovirus pneumonia, adenovirus hepatitis, and human immunodeficiency. Overall, 11 patients (22%) relapsed (4 of 33 acute myeloblastic leukemia in CR1) at a median of 235 (range 46-528) days. Mean posttransplantation follow-up was 1062 (median 560; range 10-4177) days. Thirty-three patients (66%) remained disease free at a mean of 1,118 (median 1439; range 159-4,177) days. For all patients, the performance status was between 82% and 100% (median 100). CONCLUSION: T-cell depletion with CAMPATH-1 effectively prevents GVHD, particularly the severe acute forms, without leading to excessive risk of relapse in acute leukemia.
CD59 is a complement regulatory protein and may also act as a signal-transducing molecule. CD59 transgenic mice have been generated using a CD59 minigene (CD59 minigene-1). Although this minigene contained a 4.6-kilobase pair 5'-flanking region from the human CD59 gene as a promoter, the expression levels of the CD59 mRNA were substantially lower than those observed in humans, suggesting that CD59 gene expression might also require other transcriptional regulatory elements such as an enhancer. To investigate the transcriptional regulation of the CD59 gene, we used three cell lines that express CD59 at different levels. We have identified DNase I-hypersensitive sites in intron 1 in HeLa cells, which express CD59 at high levels, but not in Jurkat (intermediate level) or Raji cells (low level). Furthermore, cell line-specific enhancer activity was detected in a fragment containing these DNase I-hypersensitive sites. The CD59 enhancer was mapped to between -1155 and -888 upstream of the 5'-end of exon 2. To investigate the enhancer activity in vivo, a new CD59 minigene was constructed by the addition of the enhancer fragment into CD59 minigene-1. High expressor CD59 transgenic mice were generated using the new minigene.
We have re-investigated the roles of CD4 and CD8 T cell subsets in skin graft rejection across a single class I MHC disparity. Recipient mice were transplanted with skin from donors transgenic for the class I MHC molecule Kb. As expected, CD8 T cells were sufficient for rapid injection; but surprisingly, CD4 T cells were also competent to do the same. Rejection was dependent on one or the other subset, since elimination of both resulted in indefinite graft survival. The possibility that alloantibody was the downstream effector of CD4 mediated rejection was excluded because CD8-depleted mice rendered B cell deficient still rejected rapidly, but T cell-depleted recipients with pre-existing high titers of alloantibody were unable to do so. In addition, if CD4 cells act to reject by recruiting and/or activating macrophages then this was not dependent on CR3, IFN-gamma or TNF-alpha. Transplantation of skin grafts where the MHC class I disparity was at the level of passenger leukocytes only, demonstrated that transient bystander damage could occur, but that this was insufficient to result in full rejection. We surmise that for CD4 T cells to reject an MHC class I-incompatible graft it is necessary that an appropriate allogeneic peptide is processed and presented in the context of recipient MHC class II. CD4 T cells from B6 mice may fail to reject skin from MHC class I mutants because of the lack of such MHC class II-restricted presentation.
Pityriasis rubra pilaris (PRP) refers to a group of erythematous, scaling dermatologic conditions that have been associated with seronegative arthritis. We report a case of polyarthritis in a young man with PRP in which magnetic resonance imaging suggested an entheseal-based pathology for the joint disease. The arthritis, but not the skin condition, demonstrated dramatic response to anti-tumor necrosis factor immunotherapy.
The elective treatment of patients with multiple sclerosis, using a humanized anti-leukocyte (CD52) monoclonal antibody (Campath-1H), has illuminated mechanisms that underlie the clinical course of the disease. Twenty-seven patients were studied clinically and by magnetic resonance imaging (MRI) before and for 18 months after a single pulse of Campath-1H. The first dose of monoclonal antibody was associated with a transient rehearsal of previous symptoms caused by the release of mediators that impede conduction at previously demyelinated sites; this effect remained despite selective blockade of tumor necrosis factor-alpha. Disease activity persisted for several weeks after treatment but thereafter radiological markers of cerebral inflammation were suppressed for at least 18 months during which there were no new symptoms or signs. However, about half the patients experienced progressive disability and increasing brain atrophy, attributable on the basis of MRI spectroscopy to axonal degeneration, which correlated with the extent of cerebral inflammation in the pretreatment phase. These data support the formulation that inflammation and demyelination are responsible for relapses of multiple sclerosis; that inflammatory mediators, but not tumor necrosis factor-alpha, cause symptomatic reactivation of previously demyelinated lesions; and that axonal degeneration, conditioned by prior inflammation but proceeding despite its suppression, contributes to the progressive phase of disability. These results provide evidence supporting the emerging view that treatment in multiple sclerosis must be given early in the course, before the consequences of inflammation are irretrievably established.