Effect of Treatment With Tabalumab, a B Cell-Activating Factor Inhibitor, on Highly Sensitized Patients With End-Stage Renal Disease Awaiting Transplantation.

B cell-activation factor (BAFF) is critical for B cell maturation. Inhibition of BAFF represents an appealing target for desensitization of sensitized end-stage renal disease (ESRD) patients. We conducted a Phase 2a, single-arm, open-label exploratory study investigating the effect of tabalumab (BAFF inhibitor) in patients with ESRD and calculated panel reactive antibodies (cPRAs) >50%. The treatment period duration was 24 weeks. Eighteen patients received tabalumab, at doses of 240-mg subcutaneous (SC) at Week 0 followed by 120-mg SC monthly for 5 additional months. Patients were followed for an additional 52 weeks. Immunopharmacologic effects were characterized through analysis of blood for HLA antibodies, BAFF concentrations, immunoglobulins, T and B cell subsets, as well as pre- and posttreatment tonsil and bone marrow biopsies. Significant reductions in cPRAs were observed at Weeks 16 (p = 0.043) and 36 (p = 0.004); however, absolute reductions were small (<5%). Expected pharmacologic changes in B cell subsets and immunoglobulin reductions were observed. Two tabalumab-related serious adverse events occurred (pneumonia, worsening of peripheral neuropathy), while the most common other adverse events were injection-site pain and hypotension. Three patients received matched deceased donor transplants during follow-up. Treatment with a BAFF inhibitor resulted in statistically significant, but not clinically meaningful reduction in the cPRA from baseline (NCT01200290, Clinicaltrials.gov).

In vitro monitoring of in vivo development of human anti-thymoglobulin antibodies by ELISA.

Thymoglobulin (rATG), polyclonal immunoglobulin, is prepared from rabbits immunized with human thymocytes. It is effective in prevention and treatment of renal allograft rejection. Human antibodies against antilymphocyte preparations can reduce efficacy by accelerating drug clearance or by inducing serum sickness. We developed an enzyme-linked immunosorbent assay (ELISA) to study posttreatment development of anti-rATG. In an Institutional Review Board-approved trial, we tested 101 allograft recipients for anti-rATG antibodies. Patients received rATG intravenously at 1.25 to 2.0 mg/kg/d for 2 to 14 days. Serum samples were obtained pretreatment and at weeks 1, 2, 4, 6, and months 3 and 6 post-rATG. ELISA plates were coated with rATG (10 microg/mL). Samples were diluted 1:100 and tested in quadruplicate. Positive samples were titrated. Horseradish peroxidase-conjugated (HRPO) affinity-purified goat anti-human immunoglobulin G (H&L) antibody reacted with bound human antibody. A chromagenic substrate for HRPO was added and optical density (OD, 490 nm) was read. An OD of twice the negative control was considered positive. Mean ODs of negative and positive controls were 0.113 +/- 0.030 and 1.042 +/- 0.196, respectively. Ten patients had detectable anti-rATG before rATG administration (1:100). Thirty-five of 101 patients (35%) developed anti-rATG antibody. Patients showed an initial positive anti-rATG antibody from days 8 to 59 after infusion and titers from 1:100 to 1:4000. In spite of rATG's postulated anti-B-cell activity, this study confirms that rATG induces sensitization at a frequency and titer seen with other xenogeneic antilymphocyte antibodies. Formation of such antixenoantibodies can have a negative impact on treatment response and hence warrant monitoring.

New crossmatch technique eliminates interference by humanized and chimeric monoclonal antibodies.

Humanized and chimeric antilymphocyte antibodies (Ab) are used to prevent and treat rejection and for treatment of human disease. Rituximab (RIT, anti-CD20), daclizumab (DAC; anti-CD25), alemtuzumab (ALE; anti-CD52), or infliximab (IFX) may interfere with Ab detection methods such as complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human Ab or fix complement and are not differentiated from anti-allo-Ab. A new enzyme-linked immunosorbent assay crossmatch (XM) utilizing class I and II HLA antigens from donor cells called Transplant Monitoring System (TMS; GTI, Waukesha, Wisc) potentially precludes interference by eliminating non-major histocompatability complex antigens. To test this, normal sera (nonsensitized volunteers) were supplemented with 0.1 or 10 microg/mL of RIT, DAC, IFX or ALE, and were tested using three methods: the TMS T-cell CDCXM with antihuman globulin (AHG); and B-cell CDCXM without AHG; and FCXM with mean channel shifts of 45 and 150 indicating positive T-cell and B-cell crossmatch, respectively. No reactivity occurred with normal sera using any crossmatch technique. At 0.1 and 10 microg/mL, RIT interfered with CDC B-cell, but not T-cell crossmatch. RIT at 10, but not 0.1 microg/mL interfered with B-cell FCXM. No interference occurred with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM but neither class I nor II TMS. DAC did not interfere with CDC or FCXM at 0.1 microg/mL, but gave false positive B-cell FCXM and CDCXM with some samples. No interference by DAC occurred using TMS. TMS may be useful to differentiate de novo donor-specific Ab after treatment with humanized or chimeric Ab.

Reduced human IgG anti-ATGAM antibody formation in renal transplant recipients receiving mycophenolate mofetil.

Exposure to the equine-derived polyclonal antithymocyte preparation, ATGAM, frequently elicits human anti-ATGAM antibody formation. The influence of concomitant immunosuppressants on this antiantibody response has not been established. We therefore evaluated IgG antibody formation to ATGAM in 47 patients receiving ATGAM as part of a prospective, randomized, double-blinded study of mycophenolate mofetil versus azathioprine for maintenance immunosuppression after primary cadaveric renal transplantation. All patients received ATGAM for induction of immunosuppression plus methylprednisolone, prednisone, and cyclosporine. In addition, patients were randomized to receive maintenance immunosuppression consisting of either azathioprine (AZA) 1-2 mg/kg/day, mycophenolate mofetil 2 gm/day (MMF2), or mycophenolate mofetil 3 gm/day (MMF3). Patient sequential sera were independently tested for IgG anti-ATGAM antibody by 2 laboratories, which were blinded to treatment arm assignments, using enzyme-linked immunosorbent assays. Both laboratories found significantly greater anti-ATGAM antibody formation in group AZA compared with groups MMF2 and MMF3: laboratory 1 reported sensitization rates in the 3 groups of 94% (AZA), 50% (MMF2) (P < 0.02 vs. AZA), and 60% (MMF3) (P < 0.05 vs. AZA); and laboratory 2 reported rates of 67% (AZA), 17% (MMF2) (P < 0.02 vs. AZA), and 10% (MMF3) (P < 0.02 vs. AZA). In addition, fewer patients formed high titer antibody in the MMF arms compared to the AZA arm: 56% (AZA), 0% (MMF2) (P < 0.02 vs. AZA), and 20% (MMF3) (P < 0.02 vs. AZA) of patients for laboratory 1; and 20% (AZA), 0% (MMF2) (P < 0.05 vs. AZA), and 0% (MMF3) (P < 0.05 vs. AZA) of patients for laboratory 2. Differences in test results between the 2 laboratories were explained by differences in the sensitivity of their respective immunoassays and in the criteria used for assigning a positive result to test specimens. In this protocol, MMF at 2-3 gm/day was associated with a reduced incidence and titer of IgG anti-ATGAM antibody formation compared with standard azathioprine dosing. Although MMF previously has been reported to inhibit T cell responses that mediate acute cellular rejection, this is the first demonstration that MMF significantly inhibits human B cell responses to antigen in vivo.

Predictive value of enzyme-linked immunosorbent assay-detected IgG anti-HLA antibodies for pediatric renal allograft rejection.

BACKGROUND: Measurement of panel-reactive antibody (PRA) with an enzyme-linked immunosorbent assay using soluble HLA class I molecules (PRA-STAT) in adult renal transplant recipients predicted graft loss and rejection. We sought to confirm this finding in pediatric recipients, an immunologically distinct group. METHODS: The population consisted of 158 renal transplants in 146 patients (age range, 1-21 years). PRA was determined with PRA-STAT and microlymphocytotoxicity (CDC), using final cross-match sera. An elevated test was defined as > or =5% reactivity. Statistical analysis for rejection used the chi-square test and for graft survival used the log-rank test. RESULTS: Thirty-five patients (22%) had %PRA-STAT > or =5%, compared with 26 (16%) with %PRA-CDC > or =5%. The percentage with elevated %PRA-STAT was found to correlate with subsequent transplantations (first, 15%; second, 67%; third, 75%). Subsequent analyses utilized only the 136 primary recipients, of whom 20 (15%) had %PRA-STAT > or =5% and 16 (12%) had %PRA-CDC > or =5%. Elevated %PRA-STAT correlated with rejection at 3 months (65% vs. 36%), 12 months (84% vs. 50%), and 24 months (84% vs. 54%) (P<0.05). No association was found between elevated %PRA-CDC and rejection. Patients with %PRA-STAT > or =5% vs. %PRA-STAT <5% had graft survival at 1 year of 89% vs. 84%, at 2 years of 88% vs. 77%, and at 3 years of 61% vs. 72% (not significant). CONCLUSIONS: Use of %PRA-STAT > or =5% identifies pediatric recipients who are at increased risk for rejection and may benefit from more potent immunosuppression and/or closer monitoring of graft function.

Comparison of Minnesota antilymphoblast globulin and Upjohn antithymocyte globulin for induction immunosuppression of human renal allografts.

BACKGROUND: An analysis of heterologous polyclonal antisera in first renal transplants was continued after replacement of Minnesota antilymphoblast globulin (MALG) with antithymocyte globulin (ATGAM), testing the hypothesis that these are functionally equivalent drugs. METHODS: Sequential induction immunosuppression used MALG (20 mg/kg/day, n = 33) or ATGAM (15 mg/kg/day, n = 14), corticosteroids, azathioprine and cyclosporine. White blood cell, platelet, and T-cell subsets were measured. Percent of patients with and time to first rejection were determined. Anti-horse antibody was measured by enzyme-linked immunosorbent assay. Minimum follow-up after transplantation was 1 year. RESULTS: Human leukocyte antigen mismatch, peak and current panel reactive antibodies, age, gender, percent cadaver donors and diabetic recipients were similar. Depletion of CD2, CD3, CD4, and CD8 T-cell subsets and platelet and white blood cells was similar. Early renal function was better with MALG than with ATGAM (p = 0.005, ANOVA), but by 2 weeks the groups were similar. The percent of patients receiving MALG versus patients receiving ATGAM with cytomegalovirus (28 versus 50), anti-horse antibodies (50 versus 62), and rejection (58 versus 50) and the median day of first rejection (48 versus 47) were similar. Three grafts were lost. CONCLUSIONS: MALG and ATGAM are equally effective in eliminating T cells and preventing and delaying the onset of renal allograft rejection.

Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies (mAb) analyzed within the T-cell group from the Second Swine CD Workshop, six mAb fell within clusters T10 and T11 (No. 088, STH164; No. 148, FY1A3; No. 149, FY2C1; No. 150, FY1H2; No. 151, FY2A11; No. 169, BB23-8E6). The mAb within these two groups gave a similar appearance on flow cytometry and stained all peripheral blood T-cells as defined by CD4 and wCD8 staining. All six mAb precipitated a 24 kDa protein. On the basis of inhibition analyses performed as part of the workshop and from published data, the mAb define at least three epitopes. There is only minimal stimulation of resting peripheral lymphocytes, but four of the mAb produce strong stimulation in the presence of PMA. With the exception of STH164, all have been shown to react with CD3 epsilon-transfected COS cells. The new mAb, therefore, react with three epitopes on porcine CD3 epsilon designated CD3a (BB23-8E6, FY2A11), CD3b (FY1A3, FY2C1), and CD3c (FY1H2). mAb STH164 appears to be reactive with another epitope, however, since its reactivity with CD3 has not been confirmed it is designated as wCD3.

Analyses of monoclonal antibodies reacting with porcine CD5: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies analyzed within the T-cell group, three mAbs fell within cluster T13 including the CD5a standard b53b7 (No. 174). The two new mAbs 1H6/8 (No. 058) and BB6-9G12 (No. 166) both precipitated 55 and 60 kDa proteins that were of similar molecular weights as the standard. Staining patterns on the various cell types were similar. Both new antibodies inhibited the binding of the CD5a reference mAbs b53b7 to peripheral lymphocytes. These mAbs, therefore both react with the CD5a epitope bringing the number of anti-porcine CD5 mAbs to eight, all of which appear to recognize the same epitope.

Analyses of monoclonal antibodies reacting with porcine wCD6: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.

Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop.

After initial evaluation of the 176 new and 19 control monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cell/activation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyer's patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number.

Differences in alloimmune response between elderly and young mice.

OBJECTIVE: The upper age of renal transplant recipients is rising on the transplant wait list. Age-dependent immune responsiveness to new antigens has not been thoroughly studied. This study used a mouse model of alloantibody response to neoalloantigen to study age-related differences. METHODS: Transgenic huCD20-C57BL/6 mice were immunized intraperitoneally with BALB/c splenocytes (2.5 × 10(7)) at baseline and 1 month. Plasma samples were collected at baseline and 1 and 2 months after inoculation, frozen, and tested in a batch run (n = 22). Samples were tested by flow cytometric crossmatch for alloantibody with 2-fold serial dilution from neat to 1:640 using BALB/c splenocytes as targets. The sum of the median fluorescence intensity of the tested sample was calculated after subtracting that of an autologous serum control. Elderly mice (ELD; 42-103 weeks) at inoculation were compared with younger mice (YOU; 11-15 weeks). Statistical analysis was performed with 2-sample t test. RESULTS: Mean age (weeks) between the groups was significantly different (ELD 69.3 ± 9.6 vs YOU 13.4 ± 1.4; P < .001). There was no difference in alloantibody between groups at baseline (ELD 0.7 ± 3.1 vs YOU 0.6 ± 0.4; P = .93). There was a higher alloantibody response at 1 month for YOU (52.9 ± 31.78) compared with ELD (5.12 ± 8.18). There was a greater difference after the 2 month (YOU 109.38 ± 66.43 vs ELD 21.97 ± 27.14; P < .0024). CONCLUSIONS: There was a difference in response to new alloantigen in this animal model. Older animals had significantly decreased responses to new alloantigen stimulation 1 month after inoculation and even more profound decreases at 2 months compared with young animals. This model may be used to study differences in immune refractoriness to antigen signaling. It may be important to adapt clinical immunosuppression in the aged population to possible decreased responses to immune stimulation.

Elimination of false-positive Histoplasma antigenemia caused by human anti-rabbit antibodies in the second-generation Histoplasma antigen assay.

False-positive Histoplasma antigenemia was reported in solid organ allograft recipients who had received rabbit anti-thymocyte globulin (RATG, RATG) caused by human anti-rabbit antibodies (HARA). A second-generation Histoplasma antigen detection assay was developed to overcome false positivity caused by HARA. With the second-generation assay, false-positive results were eliminated in 18 of 19 cases without reduction in the sensitivity in patients with histoplasmosis. In fact, sensitivity for detection of antigenuria in patients with acquired immunodeficiency syndrome and disseminated histoplasmosis was higher in the second-generation assay. Physicians should be aware of the potential for false-positive results in sandwich immunoassays in specimens from patients who have received RATG.

An evaluation of the cyclosporine parent-compound-specific whole blood TDx assay.

The new Abbott TDx cyclosporine parent-compound-specific fluorescence polarization immunoassay (TDxP) was evaluated and compared to the cyclosporine-and-metabolites-specific TDx (TDxT) and a cyclosporine parent-compound-specific radioimmunoassay (RIA) (Sandimmun-Kit, INCSTAR). The TDxP assay was linear within the range of 31 to 1600 ng/ml (r = 0.985) with a lower limit of detection of less than 31 ng/ml. The TDxP had excellent intra- and interassay reproducibility (CV = 1.2 to 4.5) that was significantly better than that of the radioimmunoassay. 230 whole blood samples obtained from 65 kidney, 19 liver, and 8 pancreas transplant recipients were analyzed with each of the three assay methods. TDxP had a much stronger correlation with the RIA than did TDxT (r = 0.95 versus 0.83). The difference between the correlations was greatest for the liver and pancreas recipients. The mean ratio of the cyclosporine level determined by TDxP to RIA was 1.0 versus 2.4 for TDxT to RIA. The new TDxP assay provides results equal to a parent-compound-specific RIA but with the added advantages of decreased sample turn-around time and improved intra- and interassay coefficients of variation.

False-positive Histoplasma antigenemia caused by antithymocyte globulin antibodies.

False-positive Histoplasma antigen results were identified in two patients who received rabbit antithymocyte globulin (ATG, Thymoglobulin(R)) to prevent allograft rejection. To determine the prevalence of false-positive results following the administration of Thymoglobulin, sequential specimens were tested from a cohort of transplant recipients. Of 107 such patients, 17 (15.9%) demonstrated false-positive tests for Histoplasma antigenemia. False antigenemia peaked at 2-4 weeks after ATG administration and cleared over the next few months. Physicians should be aware of the potential for false-positive results in specimens from patients who have received ATG.