Removal of therapeutic anti-lymphocyte antibodies from human sera prior to anti-human leukocyte antibody testing.

Both monoclonal (e.g. Orthoclone (OKT3), rituximab) and polyclonal (e.g. ATGAM, Thymoglobulin (Thymo)) anti-lymphocyte Abs (ALAs) are used extensively in organ transplantation for immunosuppression induction, desensitization, and treatment of acute rejection. ALAs often interfere with post transplant immunologic monitoring. We describe a method that uses magnetic beads to selectively remove ALAs from patient serum. Rabbit anti-mouse Fc-specific (180 mug), or rabbit anti-mouse Fab-specific (180 microg), or rabbit anti-horse heavy and light chain-specific and rabbit anti-horse F(ab')2 (200 microg) (Jackson Immunoresearch) was adsorbed to 6.7 x 10(8) Dynabeads M-280 conjugated with sheep anti-rabbit IgG (Dynal Biotech). Fifty microliters of normal human serum (NHS) with 2 microg/ml of OKT3 or 100 microg/ml ATGAM, Thymo, or rituximab were incubated with conjugated beads for several incubations. NHS containing ALAs before and after treatment by the protocol were incubated with human lymphocytes and labeled with FITC-antibody to immunoglobulin of the species used to produce the particular ALA. Residual ALA was determined using flow cytometry. Average median channel for serum with or without ALA was 11.1 and 0.120, respectively for OKT3; 64.4 and 0.344 for ATGAM; 108.5 and 0.200 for Thymo; and 1022.5 and 11.4 for rituximab. Treatment lowered the median channel for serum with OKT3 to 0.103, 0.309 for ATGAM, 0.199 for Thymo, and 12.1 for rituximab. ALAs can be effectively removed from serum by the use of magnetic beads conjugated with Ab specific for ALA thereby permitting immunologic monitoring without interference.

Rituximab for reduction of anti-HLA antibodies in patients awaiting renal transplantation: 1. Safety, pharmacodynamics, and pharmacokinetics.

BACKGROUND: Preformed HLA antibodies (Ab), reported as panel-reactive antibody (PRA), prolong patient waiting time for kidney transplantation. We hypothesized that rituximab (RTX) could reduce PRA via B-cell depletion. This initial study reports the safety, pharmacokinetics, and pharmacodynamics of RTX in patients with end-stage renal failure. METHODS: The study was an investigator-initiated single-dose, dose-escalation phase I trial of RTX in chronic dialysis patients (PRA >50%). It was approved by the Institutional Review Board and the Food and Drug Administration. Nine subjects were treated with a single dose of RTX (n=3 per group) at 50, 150, or 375 mg/m. Peripheral lymphocyte cell surface markers and HLA Ab levels (%PRA and titers) were tested using flow cytometry. RESULTS: There were four significant adverse events: a suspected histoplasmosis infection; two Tenchkoff dialysis catheter infections; and fever (38.7 degrees C) during infusion. At 2 days after RTX therapy, there was depletion of CD19 cells (pre-RTX 181+/-137 vs. post-RTX 12+/-5.6, P =0.006). In 2 (22%) of 9 subjects, there was no appreciable change in PRA. Among the other seven patients, one had a decrease in PRA from 87% to 51% with a concurrent decrease in fluorescence intensity; five patients had changes in histogram architecture suggesting loss of antibody specificity; and one patient had a fourfold decrease in PRA titer from 1:64 to 1:16 at 6 months after treatment. In addition, one of the seven patients converted a donor-specific crossmatch to negative and underwent a successful living donor kidney transplantation. CONCLUSIONS: RTX can be safely administered and may be an effective agent to reduce high-titer anti-HLA Abs in subjects awaiting kidney transplantation.

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab.

Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F(c) receptors removes CD20 from B cells. After CD20 is removed, RIT should not bind, making it possible to detect class I or class II antibodies on treated B cells. In this study, we incubated RIT with normal human serum (NHS, negative control) or pooled sera from highly sensitized (>50% panel reactive antibody, HLA+) subjects awaiting renal transplantation (positive control) and then performed B-cell flow cytometric crossmatches using untreated or pronase treated B cells as targets. We observed that untreated B cells incubated with RIT-spiked NHS displayed a significant increase in surface fluorescence compared with NHS without RIT, similar to the fluorescence that occurs with a positive crossmatch. In contrast, when CD20 was cleaved from the B cells with pronase, B cells displayed a negative crossmatch with the RIT-spiked NHS. In addition, there was no change in the crossmatches of pooled high panel reactive antibody (PRA) sera after pronase treatment. RIT could be used without worry about losing the ability to perform transplant immunologic monitoring.

In vivo human B-cell subset recovery after in vivo depletion with rituximab, anti-human CD20 monoclonal antibody.

Rituximab, chimeric anti-human CD20 monoclonal antibody approved for B-cell lymphoma, depletes circulating B cells. Little data exist on its use for nonmalignant diseases or B-cell subset recovery after treatment. We hypothesized that rituximab may reduce panel reactive alloantibodies (PRA) in dialysis patients awaiting renal transplantation and performed a single-dose, dose-escalation phase 1 trial. Here we report changes in lymphocyte phenotypes in subjects treated with rituximab alone. Nine subjects, 44 +/- 10 years(Yr); (5 F, 4 M) received one dose (n=3) at 50, 150, or 375 mg/m(2) without other immunosuppression. Blood was collected before dosing and intervals thereafter. No significant changes in leukocytes, total or CD3(+) lymphocytes were noted. In all, there was CD19(+) depletion by day 2(D2) (12.0 +/- 5.6 cells/mm(3) vs. 181 +/- 137, D0; p<0.01) and CD20(+) (11.0 +/- 12.0 vs. 205 +/- 116, D0; p<0.01). At 6 months (mo), CD19(+) and CD20(+) remained low (51.1 +/- 42.2, p<0.05; 75.4 +/- 38.7, p<0.05, respectively) compared to D0. CD19(+)CD5(+) cells recovered more rapidly, returning to baseline by 6mo while B memory cells (CD19(+)CD27(+)) remained low (32.3 +/- 29.0) at 1Yr (7.5 +/- 4.5; p<0.001) and 2Yr (12.1 +/- 7.9; p<0.001) after treatment. We conclude that single dose rituximab ablates B cells in high PRA dialysis patients awaiting transplantation. B-cell ablation, particularly memory B cells, was long-lasting, lagging repopulation by CD5(+) B cells.

Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phiX174.

The response to primary immunization in patients treated with Rituximab (RIT) is not clear. We studied the in vivo antibody response of chronic renal failure (CRF) patients to the neoantigen bacteriophage phiX174 given alone or after ablation with RIT. Eighteen CRF subjects received two immunizations with phiX174 separated by 6 weeks. Nine subjects received a single dose of RIT. The intensity and immunoglobulin isotype of the antibody response (K(v)) were measured post-infusion. In addition, three subjects previously immunized and treated with RIT underwent a third and fourth immunization with phiX174 and a tetanus control 2 years later. RIT significantly decreased peak K(v) responses when compared to both historic non-CRF controls and to CRF subjects. CRF itself decreased peak K(v) responses compared to non-CRF controls. Percent-ratio of anti-phage IgM to IgG was significantly decreased in RIT treated subjects. One of three subjects treated with RIT was found to have developed a partial B cell tolerance to phiX174 administration 2 years later. RIT decreases antibody production and isotype switching to neoantigens and might be useful to prevent antibody response to therapeutic drugs and to newly transplanted organs.