Efficacy of IVIG for Treatment of De-Novo Donor Specific Antibodies in Renal Transplant Recipients

Abstract Development of Donor Specific Antibodies (DSA) is linked to worsened outcomes in renal transplant recipients. Intravenous immunoglobulin (IVIG) is an immune-modulator utilized in treatment of antibody mediated rejection. A retrospective review of kidney transplant cases at Lehigh Valley Health Network (LVHN) from January 2009 to June 2014 was performed to evaluate the efficacy of IVIG in treatment of new DSAs. All patients undergoing renal transplant at LVHN are cross-match compatible at the time of procedure. Desensitization is not utilized. All highly sensitized patients (PRA\u3e50%) receive prophylactic IVIG monthly x 4 months post-transplant. Study patients that tested positive for DSA post-transplant were treated with additional IVIG (0.5g/kg monthly). 95 patients were treated with IVIG during the study period. Of 55 patients with newly positive DSA, 24 of these cleared the DSA after IVIG treatment. IVIG was more effective in clearance of class I than class II DSA. Highest rates of graft loss occurred in patients that tested positive for both a class I and class II DSA. Conclusion: 1. IVIG can be a useful in eliminating DSA post-renal transplant. 2. IVIG is effective in eliminating Class I DSA. 3. Class II DSA can be difficult to eliminate and requires further investigation. Background Intravenous immunoglobulin (IVIG) is a medication that has emerged as a useful tool in modulating immunity, treatment of antibody mediated rejection (AMR), and in desensitization protocols. IVIG serves as a mediator of the immune system and as a regulator of inflammation. DSAs specifically target the transplant donor organ. The presence of DSAs has been linked to significantly worse graft survival. In patients with AMR, therapy with IVIG paired rituximab and plasmapheresis (PP) can increase graft survival by more than 40% and suppress DSAs. (1) Studies have still not demonstrated optimal regimens for treatment of new DSA. The purpose of this study was to investigate the effectiveness of IVIG in elimination of DSAs. Methodology A retrospective chart review was performed of all kidney transplant patients at Lehigh Valley Health Network who were treated with IVIG post transplantation from January 2009 to June 2014. All patients were cross match compatible (no DSA presence at time of transplant) and had not undergone desensitization protocols. All highly sensitized patients (PRA greater than 50%), were prophylactically treated with 0.5g/kg of IVIG monthly for four doses beginning at the time of transplant per the center’s protocol. These patients had monthly DSA tests for at least one year. Nonsensitized patients (PRA less than 50%) did not receive prophylactic IVIG. These patients were checked for DSAs after 1, 6, and 12 months and when symptoms arose (elevated creatinine, proteinuria). Patients that tested positive for DSA post-transplant were treated monthly with 0.5g/kg of IVIG until clearance of DSA. These patients also underwent renal transplant biopsy at discovery of DSA. Successful treatment was considered elimination of DSA for greater than 1 month. Time to DSA clearance, dates of graft loss, AMR, and development of Class I versus Class II DSA were noted. Results Of the 293 patients who received kidney transplantation at Lehigh Valley Health Network in the study period, 95 were treated with IVIG. 55 patients that were treated with IVIG had a positive DSA (57.8%). 24 (43.6%) of those patients cleared the DSA after treatment with IVIG. Of the remaining 31 patients, 28 did not clear the DSA and 3 patients expired during the study interval. 61 of the 95 (64.2%) patients received prophylactic IVIG. Out of those, 41/61 (67.2%) never developed a DSA while 20/61 (32.8%) did. Out of the 20 that developed a DSA, 9 cleared the DSA. 34 non-sensitized patients (12% total non-sensitized transplants) developed DSAs and then received treatment with IVIG. Outcomes Sensitized versus Non-sensitized Patients Treated with IVIG Outcomes of Class I versus Class II DSAs Treated with IVIG IVIG was more effective in the clearance of class I DSA’s than class II. Following treatment with IVIG, 8/12 (66.7%) of the Class I DSAs cleared and 11/26 (42.3%) of the class II DSAs cleared (p=0.05). Clearance rates were lower in patients starting with both Class I and II antibodies, but Class I cleared more frequently. In only one case, the patient cleared the class II DSA first, however this patient expired only two months later while the class I was still present. Lowest rates of graft loss occurred in patients with a class I DSA, followed by class II, and lastly if a patient had both (p=0.02). P=0.020 P=0.05 Conclusions 1. IVIG can be a useful in eliminating DSA post-renal transplant. 2. IVIG is effective in eliminating Class I DSA. 3. Presence of Class II DSA is associated with higher rates of graft loss. Class II DSA can be difficult to eliminate and requires further investigation, possibly in combination with biologic agents. 4. Limited numbers of patients at this time make statistically significant results difficult to achieve. Further investigation is necessary. References Shehata et al. (2010) The use of immunoglobulin Therapy for Patients Undergoing Solid Organ Transplantation: an Evidence- based practice guideline. Transf Med Rev 2010; 24:7-27. Rocha P et al. (2003) Beneficial Effect of Plasmapheresis and Intravenous Immunoglobulin on Renal Allograft Survival of Patients with Acute Humoral Rejection. Transplantation 75:1490-1495 Lefaucher et al. (2009) Comparison of Combination plasmapheresis/IVIG/Anti-CD20 Versus High Dose IVIG in the Treatment of Antibody-Mediated Rejection. Am J Transplant 9:1099-110

Sonographic Findings in Acute Uterine Inversion

We present a case of acute uterine inversion in the third stage of labor in which critical management decisions were facilitated by ultrasound imaging in the operating room. Identification of the ovary and adnexa pulled into the inclination of the inversion allowed successful diagnosis and guidance of uterine replacement

Determination of reduced disulfide groups in monoclonal antibodies.

Reduction of disulfide bonds to sulfhydryl groups for direct radiolabeling of antibodies for immunoscintigraphic and therapeutic applications continues to be of considerable interest. Sensitive spectrophotometric methods have been evaluated that will enable investigators to determine submicrogram quantities of cysteine units produced, for the assurance of controlled reduction. One method, which generates a cysteine-ninhydrin complex (520 nm), has a molar extinction coefficient of 30 250 and can determine 0.04 micrograms/ml cysteine units with an absorbance of 0.01. The method has been applied to determine the quantity of cysteine groups produced by the reduction of an immunoglobin G antibody with five different reducing agents in normal to five times the previously determined optimal molar ratios. The quantities of cysteine units produced from the controlled reduction from 240 micrograms immunoglobin G ranged from 0.073 +/- 0.01 to 1.07 +/- 0.04 micrograms, which were merely 0.54 +/- 0.08% to 7.9 +/- 0.28% of the total available disulfide groups in the protein

Technetium-99m-labeled monoclonal antibodies for immunoscintigraphy. Simplified preparation and evaluation.

Ascorbic acid incubated with monoclonal antibodies (22 degrees C, 60 min, pH 6.5) at a molar ratio of 3500:1, reduced 2.7 +/- 0.2% of the available disulfides to sulfhydryl groups that strongly bind 99mTc, and provided greater than 95% labeling efficiency for several IgM, IgG and F(ab\u27)2 antibodies. The colloid formation was consistently less than 3% and the stability of the tracer when challenged with DTPA and cysteine was excellent. The immunospecificity of labeled antibodies as determined by immobilized specific antigen assay was 84 +/- 1% for IgM and 82.6 +/- 1.1% for IgG antibodies. For in vivo evaluation in mice bearing experimental abscesses and tumors, corresponding 125I-labeled antibodies served as controls. The liver uptake was similar (P = 0.76 and P = 0.12) for 99mTc or 125I labeled antinuclear antibody TNT-1 in mice bearing abscesses as well as for 99mTc-TNT-1-F(ab\u27)2 and 125I-TNT-1-F(ab\u27)2 in mice bearing tumors. Higher but statistically insignificant (P = 0.08, 0.18, and 0.73) urinary excretion was noted for 99mTc-antibodies. For corresponding 99mTc- and 125I-labeled antibodies, the abscess to muscle ratios (3.3 +/- 0.5 vs. 3.4 +/- 0.8) and tumor to muscle ratios (10.04 +/- 4.4 vs. 10.54 +/- 3.0) were similar. The high 99mTc-TNT-1-F(ab\u27)2 uptake permitted excellent scintigraphic visualization of tumors whereas the nonspecific 99mTc-HSA did not (tumor/muscle ratio: 2.4 +/- 0.3). This method is simple, reliable, and adaptable to an instant labeling technique

Technetium-99m labeled monoclonal antibodies: evaluation of reducing agents.

We have evaluated five compounds, stannous chloride (SnCl2), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with 99mTc. The reduction of 99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 +/- 1% for an IgM and 82.6 +/- 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such 99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no 99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 +/- 2.9% per gram for 125I-TNT-1 (IgG) and 6 +/- 5.1% per gram for the same MoAb labeled with 99mTc using AA. The AA technique promises to label antibodies with 99mTc and perhaps with 186Re, by a simple kit procedure

Rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: preparation and evaluation.

Rhenium-186 has been determined to be a leading radionuclide for radioimmunotherapy. However, the use of 186Re has been limited due to the lack of a convenient and efficient method by which the radionuclide can be bound to monoclonal antibodies. We have developed a simple technique to label IgM, IgG, fragmented antibodies and tumor necrosis factor-alpha with 186Re. This technique uses ascorbic acid (AA) for controlled reduction of antibody disulfide groups to sulfhydryls and SnCl2 in citric acid for the reduction of 186ReO4-. The labeling yields as determined by instant thin-layer chromatography, molecular filtration and gel filtration were greater than 95% and the colloid formation was less than 5%. The labeled antibodies were stable when challenged with 100 and 250 molar excess of DTPA and HSA for 24 hr at 37 degrees C. SDS-PAGE analysis and autoradiography of labeled IgM, IgG and F(ab\u27)2 monoclonal antibodies indicated uniform labeling and that no fragmentation of the monoclonal antibodies had taken place during the labeling procedure. Immunospecificity of 186Re-labeled human neutrophil specific IgM, as determined by in vitro antigen excess assay, was comparable to that of indium-111-labeled c-DTPA-IgM and technetium-99m-labeled-IgM. A nuclear histone specific 186Re-TNT-1-F(ab\u27)2 was evaluated in mice bearing experimental tumors. The tumor/muscle ratios at 4 and 24 hr were 5.9 +/- 0.21 and 13.8 +/- 6.7, respectively compared to that of 2.4 +/- 0.3 at 4 hr p.i. with a nonspecific protein. The labeling technique is simple, reliable and has already been adapted to a single-vial kit preparation

Evaluation of biological response modifiers in the enhancement of tumor uptake of technetium-99m labeled macromolecules. A preliminary report.

Imaging tumors with radioactive monoclonal antibodies remains attractive but continues to be challenging. With the hypothesis that the use of biological response modifiers (BRMs) may augment the tumor uptake, technetium-99m(99mTc)-labeled tumor necrosis factor (TNF) and nuclear histone specific TNT-1-F(ab\u27)2 were evaluated in tumor bearing mice given a single dose of interferon (IFN). Ukrain or pokeweed mitogen as BRMs. As early as 1.5 h post injection (p.i.) of the radioactive macromolecules, the absolute tumor uptake (% administered dose/g) of each agent was enhanced (e.g., TNF, control = 1.8 +/- 0.4, Ukrain = 3.2 +/- 0.5, P = 0.006) and tumor to muscle ratios were elevated (e.g., TNF, control a 4.1 +/- 2.2, interferon 8.3 +/- 2.7, P = 0.01). The absolute tumor uptake remained practically unchanged at 4 h p.i. Generally with BRMs, the blood clearance was rapid and tumor/blood ratios and tumor/muscle ratios were higher than in the control group, increasing to greater than 200% for IFN as a BRM. The early enhancement in tumor uptake of macromolecules, leading to excellent delineation of tumors by scintigraphy is highly encouraging and warrants further studies to explore the full potential of BRMs