Philippe Pouletty

Results of the double-blind, randomized, multicenter, phase III clinical trial of thymoglobulin versus Atgam in the treatment of acute graft rejection episodes after renal transplantation

BACKGROUND Thymoglobulin, a rabbit anti-human thymocyte globulin, was compared with Atgam, a horse anti-human thymocyte globulin for the treatment of acute rejection after renal transplantation. METHODS A multicenter, double-blind, randomized trial with enrollment stratification based on standardized histology (Banff grading) was conducted. Subjects received 7-14 days of Thymoglobulin (1.5 mg/kg/ day) or Atgam (15 mg/kg/day). The primary end point was rejection reversal (return of serum creatinine level to or below the day 0 baseline value). RESULTS A total of 163 patients were enrolled at 25 transplant centers in the United States. No differences in demographics or transplant characteristics were noted. Intent-to-treat analysis demonstrated that Thymoglobulin had a higher rejection reversal rate than Atgam (88% versus 76%, P=0.027, primary end point). Day 30 graft survival rates (Thymoglobulin 94% and Atgam 90%, P=0.17), day 30 serum creatinine levels as a percentage of baseline (Thymoglobulin 72% and Atgam 80%; P=0.43), and improvement in posttreatment biopsy results (Thymoglobulin 65% and Atgam 50%; P=0.15) were not statistically different. T-cell depletion was maintained more effectively with Thymoglobulin than Atgam both at the end of therapy (P=0.001) and at day 30 (P=0.016). Recurrent rejection, at 90 days after therapy, occurred less frequently with Thymoglobulin (17%) versus Atgam (36%) (P=0.011). A similar incidence of adverse events, post-therapy infections, and 1-year patient and graft survival rates were observed with both treatments. CONCLUSIONS Thymoglobulin was found to be superior to Atgam in reversing acute rejection and preventing recurrent rejection after therapy in renal transplant recipients.

Correlation of ELISA-detected IgG and IgA anti-HLA antibodies in pretransplant sera with renal allograft rejection.

The present study compared the occurrence of rejection episodes during the first twelve posttransplant (Tx) months and the 1-, 2-, and 3-year graft survivals among recipients stratified by the percent panel reactive antibody (% PRA) of pre-Tx sera as detected using either an antihuman globulin determined PRA (AHG-% PRA) or an ELISA methodology detecting IgG reactive against soluble HLA class I antigens (% PRA-STAT). There was a significant correlation between AHG-PRA greater than or equal to 10% and a PRA-STAT greater than or equal to 10% (P<0.001). However, among 200 sera displaying an AHG-PRA greater than or equal to 10% (mean 57 +/- 2l%), only 69% (138/200) displayed a PRA-STAT greater than or equal to 10%. With further study the discrepant finding, of 62 sera that were AHG-PRA greater than or equal to 10% but PRA-STAT <10%, was due to the presence of IgM and/or IgG non-MHC reactivity. In contrast, among 293 sera displaying an AHG-PRA < 100% (mean 3 +/- 2%), 15% (43/293) displayed a PRA-STAT greater than or equal to 10%. There was no correlation between AHG-% PRA and rejection episodes occurring during the first twelve post Tx months. In contrast, however, there was a highly significant correlation between PRA-STAT greater than or equal to 10% and the occurrence of rejection episodes during the first twelve post-Tx months (P < 0.001). Patients with PRA-STAT greater than of equal to 10% experienced a 70% rejection frequency compared with the 35% rejection frequency for patients with PRA-STAT sera < 10% (P<0.001). A significant correlation was observed between the presence of IgG-1 and rejection (P<0.01) but not IgG-subclasses 2, 3, or 4. Of particular interest was the observation in 11 patients that the presence of ELISA-detected IgA anti-HLA class I antigen (ELISA-IgA PRA greater than or equal to 10%) was associated with a significantly reduced rejection risk compared with sera where only PRA-STAT greater than or equal to 10% was present (27% vs. 70% incidence of rejection episodes, P<0.01). Finally, patients displaying pretransplant PRA-STAT results < 10% experienced significantly improved l-, 2-, and 3- year graft survivals of 85% vs. 74%, 82% vs. 70% and 81% vs. 67%, respectively (P<0.01 for each time point), compared with patients displaying PRA-STAT results greater than or equal to 10%. These data suggest that the use of the ELISA methodology to detect IgG reactivity against soluble HLA class I antigens (PRA-STAT) may allow for the determination of a more clinically informative % PRA than the AHG-% PRA. Moreover, the presence of ELISA-detected IgA anti-HLA may act to inhibit rejection mechanisms associated with ELISA-detected IgG anti-HLA greater than or equal to 10%.

Prolongation of allogeneic heart graft survival in rats by administration of a peptide (a.a. 75-84) from the α1 helix of the first domain of hla-b7 01

Allospecific T lymphocytes mediate graft rejection through specific, direct or indirect, recognition of processed determinants of foreign MHC class I molecules. Small synthetic peptides derived from highly conserved sequences of the alpha 1 helix of the first domain of certain MHC class I molecules have been shown to inhibit CTL responses in vitro and to prolong graft survival in rats when combined with subtherapeutic doses of cyclosporine. Here, we report that the survival of LEW.1W heart allografts was significantly prolonged when transplanted into congenic LEW.1A recipients treated only with a peptide corresponding to residues 75-84 of the human HLA-B7-01 molecule (B7.75-84) before transplantation. The experimental value for mean survival time (+/- SD) in untreated recipients was 13 +/- 6 days and in peptide-treated recipients was 42 +/- 27 days (P < 0.002). A total of 64% of treated recipients had a functioning graft at 30 days, while grafts were rejected in all rats belonging to the control group within this time. Within graft-infiltrating leukocytes (GIL) in B7.75-84-treated animals, the proportion of T cells was significantly lower and that of CD5-/TCR alpha beta-/CD16-/CD8+ and MHC class II+ cells concomitantly increased, as compared with nontreated animals. GIL from B7.75-84-treated animals also exhibited a dramatic decrease (approximately 70%) of allospecific and spontaneous (NK) cytotoxic activity, whereas their proliferation and IL-2 production were similar in both experimental groups. The IFN-gamma, IL-2, and IL-10 mRNA levels from GIL from peptide-treated recipients were similar to levels of controls, reflecting a state of activation of GIL. Perforin and granzyme A mRNA, the level of which may be modulated parallel to impaired cytotoxic functions, were at similar levels in both experimental groups. These data demonstrate that B7.75-84 significantly prolongs graft survival in LEW.1A rats when given as a single agent and suggests that a specifically decreased cytotoxic response (allospecific and spontaneous) plays a major role.

Prolongation of skin allograft survival in mice following administration of ALLOTRAP.

Recently, Clayberger et al. demonstrated that ALLOTRAP, small synthetic peptides derived from a conserved region of the alpha 1 helix of certain HLA class I molecules, inhibited human CTL responses in vitro. In rats, ALLOTRAP 07 therapy combined with a subtherapeutic dose of cyclosporine led to the permanent acceptance of heart allografts. In the present study, the effect of ALLOTRAP on the survival of skin allografts in mice was studied. The tail skin of male C57B1/6 (H-2b) mice was grafted on the back of male CBA (H-2k) recipients. In untreated animals, the skin graft was rejected after 11.6 +/- 1.13 days (MST +/- SD). Cyclosporine administered orally for 5 days after transplantation prolonged graft survival to 13.1 +/- 2.13 days. ALLOTRAP 2702 prolonged graft survival to 16.57 +/- 2.15 days when administered orally for five days posttransplantation and to 18.86 +/- 0.38 when administered intraperitoneally until rejection. Thus, ALLOTRAP peptides derived from human MHC class I sequences, in addition to inhibiting human T cell responses in vitro, also prolong allograft survival in rats and mice.

Detection of panel-reactive anti-HLA class I antibodies by enzyme-linked immunosorbent assay or lymphocytotoxicity: Results of a blinded, controlled multicenter study

A soluble HLA ELISA for the detection of anti-HLA class I IgG antibodies was developed and compared to complement-dependent microlymphocytotoxicity. ELISA plates were coated with a panel of sHLA class I antigens isolated from the culture supernatants of 46 different EBV-transformed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Absorbance was read using an ELISA plate reader and assay results were analyzed by computer. Antibody specificities were determined by Fishers exact test tail analysis. The reproducibility of ELISA assay results was evaluated in a blinded, controlled multicenter study. A total of 102 serum specimens from patients on waiting lists to receive kidney transplants were tested five times by ELISA in five different laboratories. The correlation coefficients (r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and the average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint titration of several serum specimens demonstrated equivalent sensitivity of ELISA and microlymphocytotoxicity (using the anti-globulin antibody protocol). Most of the antibody specificities determined by ELISA were in agreement with specificities determined by microlymphocytotoxicity. To evaluate the correlation of ELISA and microlymphocytotoxicity (CDC) assay results the same 102 specimens were tested six times by CDC in five different laboratories. The interlaboratory correlation coefficient (r) of %PRA values determined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the average agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus microlymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This demonstrates that the detection of anti-HLA class I antibodies by soluble HLA ELISA is a reliable alternative to microlymphocytotoxicity testing.

Utility of standardized histological classification in the management of acute rejection

BACKGROUND Standardized histological grading of transplant kidney biopsies has become a primary criterion for diagnosis of rejection in immunosuppression clinical trials. METHODS A consortium of 19 transplant centers from North America, Europe, and Australia convened in 1995 to examine kidney transplant rejection. Data from the 1995 Efficacy Endpoints Conference were examined for frequency of adoption of Banff schema. Biopsy grading was correlated with clinical parameters of rejection and therapy response. RESULTS Histological confirmation of rejection episodes occurred in 73% of 953 cases, with Banff criteria adoption increasing in frequency between 1992 and 1995. Banff grading significantly correlated with clinical rejection severity (rejection creatinine: grade I, 2.8+/-0.2 mg/dl; grade II, 3.5+/-0.2 mg/dl; grade III, 4.1+/-0.3 mg/dl; P < 0.001), although nadir creatinines were similar. Response rates of Banff grades I and II to steroid therapy were not different, but only 42% of grade III rejections responded to steroids (P < 0.003. Banff grading also correlated with postrejection creatinine, day 15: grade I, 2.2+/-0.2 mg/dl; grade II, 3.0+/-0.2 mg/dl; grade III, 3.8+/-0.4 mg/dl (P < 0.001), and day 30: grade I, 2.1+/-0.1 mg/dl; grade II, 2.2+/-0.2 mg/dl; grade III, 2.7+/-0.2 mg/dl (P < 0.06). Banff grade III correlated with reduced graft survival at 1 year: grade I, 86%; grade II, 88%; grade III, 70% (P < 0.01). CONCLUSIONS This multicenter review of rejection severity confirms that standardized histologic classifications such as the Banff schema provide a reliable means for stratifying patient risk of treatment success or failure. These data support the use of Banff criteria in clinical trial design.

Soluble HLA antigens and ELISA--a new technology for crossmatch testing.

A soluble HLA ELISA for the detection of donor specific anti-HLA class I IgG antibodies was developed and compared with microlymphocytotoxicity. Donor sHLA was prepared from donor blood or purified blood lymphocytes and captured onto monoclonal antibody coated ELISA plates. After incubation of captured HLA with test serum, bound IgG antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Serum samples from patients on waiting lists to receive kidney transplants were tested by lymphocytotoxicity (AHG protocol) and/or sHLA ELISA in four different laboratories using HLA preparations from eight organ donors. Concordant crossmatch results were obtained for 854 (99%) of 864 ELISA crossmatches. In contrast, concordant results were obtained for 234 (91%) of 256 lymphocytotoxicity crossmatches. Interlaboratory reproducibility of ELISA results was 99%. In contrast, interlaboratory reproducibility of lymphocytotoxicity assay results was 78%. Endpoint titrations of serum specimens containing anti-HLA antibodies demonstrated equivalent sensitivity of ELISA and AHG lymphocytotoxicity crossmatch and similar sensitivity of ELISA and flow cytometry crossmatch. Specimens tested positive by lymphocytotoxicity without DTT treatment but negative with DTT treatment were tested negative by ELISA. Comparison of lymphocytotoxicity and ELISA crossmatch results showed an agreement of 94%. This demonstrates that detection of anti-donor HLA class I antibodies by ELISA is a reliable alternative to microlymphocytotoxicity testing.

Decreased cytotoxic activity of natural killer cells in kidney allograft recipients treated with human HLA-derived peptide

Peptides derived from a conserved region (aa 75-84) of HLA class I, overlapping the supertypic HLA-BW4/BW6 antigen region, have been shown to exhibit nonallele restricted immunosuppressive properties in rats and mice, prolonging survival of major histocompatibility complex-mismatched allografts. Furthermore, HLA-B7 peptides inhibit alloreactive cytotoxic cells, and both HLA-B7 and HLA-B2702 peptides inhibit natural killer (NK) cytotoxicity in vivo. In this article, we report on a randomized, controlled study of the safety and pharmacokinetics of HLA-B2702-derived peptide in human recipients of a first kidney allograft. Escalating doses of HLA-B2702 were compared with doses of placebo controls. No toxicity and no immunization against the peptide were noted. Although the study was not designed as an efficacy trial, patients who received the high-dose protocol (7 mg/kg) did experience more rejection episodes, but this was not statistically significant when compared with control patients. Interestingly, in human recipients, as previously observed in rodents, administration of the peptide was associated with a statistically significant decrease in the cytotoxicity of NK cells against K562 targets (P<0.001). As these peptides correspond to a region of the HLA class I molecule that interacts with the newly described NK receptors for class I, their mode of action through interaction with such receptors is discussed. As a peptide of the same sequence from HLA-B7 blocks both NK and alloreactive T cell cytotoxicity, it is possible that, in humans too, both types of cytotoxic cells are affected by this peptide. The biological significance of these observations should be confirmed in future controlled studies with a larger patient population.

Pretransplant rejection risk assessment through enzyme-linked immunosorbent assay analysis of anti-HLA class i antibodies

Soluble HLA enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-HLA class I immunoglobulin G (IgG), IgG1, IgG2, IgG3, IgG4, IgM, and IgA antibodies were developed and used to analyze retrospectively the correlation between pretransplant allosensitization and posttransplant rejection episodes in renal allograft recipients. Enzyme-linked immunosorbent assay plates were coated with 46 different soluble HLA preparations representing 40 different HLA class I antigens. After incubation with a serum specimen, bound antibodies were detected with a peroxidase-conjugated antibody. Serum specimens from 85 patients were analyzed. All patients tested positive by microlymphocytotoxicity (ie, >5% panel-reactive antibody [PRA]). Approximately half (56%) of the patients had experienced one or more rejection episodes within 12 months posttransplantation. Fifty-five patients tested positive by ELISA (total IgG %PRA >10%). A strong correlation between first-year rejection and ELISA-detected anti-HLA class I IgG1 was observed (P = 0.0004). The predictive value for IgG1 and first-year rejection was 77.5%, demonstrating that ELISA results identify patients at high risk of rejecting the transplanted kidney. Anti-HLA class I total IgG detected by ELISA also correlated with first-year rejection episodes (P = 0.04). The presence of anti-HLA class I IgG2, IgG3, IgG4, or IgM was not predictive of first-year rejection episodes. Anticlass I IgA antibodies were only found in combination with anti-class I IgG1 antibodies.

Targeting of antihapten antibodies to activated T cells via an IL-2- hapten conjugate prolongs cardiac graft survival

Antihapten antibodies binding to ligand-hapten conjugates are able to mediate complement mediated lysis in vitro. Based on this observation we propose a new in vivo immunotherapy using molecules that combine a low molecular weight hapten binding to antibodies preexisting in serum and a cell specific ligand. The ligand-hapten conjugates are potential cytotoxic drugs which may (1) be specific for a given target cell, (2) be nonimmunogenic, (3) be of low molecular weight, (4) form soluble complexes with preexisting antibodies resulting in prolonged half life of the drug, and (5) induce a potent antibody mediated rejection of target cells. These novel compounds could be useful for the elimination of certain cell subsets involved in allograft rejection, cancers, infectious diseases, etc., without some of the pitfalls of conventional immunotherapies. The feasibility of this approach was demonstrated in an animal model using a compound consisting of one interleukin 2 and one fluorescein molecule (IL-2-FITC). BALB/c mice (H2d) previously immunized and expressing anti-FITC antibodies were transplanted with a fully mismatched C57BL/6 (H2b) heterotopic heart allograft. Untreated controls rejected their graft by day 9 (MSD = 9 +/- 0.7). Mice with preexisting anti-FITC antibodies treated with IL-2-FITC maintained their grafts for 38.7 +/- 7.1 days (P < 0.02). No prolongation of graft survival was observed in immunized animals that were treated with IL-2 alone (MSD = 10 +/- 1.4). Nonimmunized animals treated with IL-2-FITC rejected their grafts on day 9.4 +/- 1.1. This demonstrates that IL-2-FITC therapy specifically prolonged graft survival in animals with circulating anti-FITC antibodies. The data suggest that a ligand/hapten pair can redirect preexisting antihapten antibodies toward target cells in vivo. Such compounds may be developed for human use as alternatives to polyclonal or monoclonal antibody therapy.