Terasaki Institute: Innovating Personalized Health through Convergent Science and Bioengineering.

The Terasaki Institute for Biomedical Innovation is an independent non-profit organization and uses an integrative approach to innovate personalized health. The Institute draws on its scientific, entrepreneurial, and engineering skill set to transform ideas into clinically applicable technologies.

Validation and cross-reactivity pattern assessment of monoclonal antibodies used for the screening of donor-specific IgG antibody subclasses in transplant recipients.

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1, and 2 µg of protein per 106 beads), and the PE-conjugated mAbs were titrated from 0.04 µg/mL to 5 µg/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform. Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1-4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1-4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1-4 subclass protein could be identified. The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation in IgG subclass DSA screening in allograft recipients.

Risk Stratification of Human Leukocyte Antigen Class II Donor Specific Antibody Positive Patients by Immunoglobulin G Subclasses.

BACKGROUND: Human leukocyte antigen (HLA) antibodies are a major cause of graft loss in mismatched transplant recipients. However, the time to graft loss resulting from antibody induced injury is unpredictable. The unpredictable nature of antibodies may be related to the subclass of antibodies. In this study, HLA immunoglobulin G (IgG) subclasses were investigated to determine whether a unique IgG subclass composition could better identify those patients at eminent risk for graft loss. METHODS: The serial serum samples from the 57 patients with post-transplant HLA class II donor specific antibodies (DSA) were tested for the three IgG subclasses (IgG1, IgG3, and IgG4). RESULTS: IgG3 and IgG4 were highly prevalent in failed patients compared to functioning patients (82 % vs. 34%, 45% vs. 20%, respectively). IgG3 development showed a distinct subclass trend between failed and functioning patients with poor graft survival (log rank p=0.0006). IgG1 was almost equally abundant in both groups (100% and 97%, respectively). Of the 5 patterns of IgG subclass combinations observed, IgG1+3+ showed the strongest association with graft failure (hazard ratio 3.14, p=0.007). CONCLUSION: Patients with IgG3 subclass HLA DSA showed lower graft survival. Post-transplant monitoring for IgG subclasses rather than total IgG monitoring may identify patients at risk for graft failure.

Anti-HLA-E monoclonal antibodies reacting with HLA-la and lb alleles like IVIg as potential IVIg-immunomimetics: an evolving therapeutic concept.

Healthy individuals have natural antibodies (Abs) reacting to allo-human leukocyte antigen (HLA)-la. This could be due to the presence of anti-HLA-E immunoglobulin G (IgG), which was revealed to recognize the peptides shared between HLA-E and HLA-la after peptide inhibition assay. Sera or plasma of multiple donors are pooled to prepare intravenous immune globulin (IVIg). The HLA-la-reactivity of IVIg is abolished when the anti-HLA-E Abs are depleted from the IVIg, suggesting that IVIg contains anti-HLA-E Abs with HLA-la reactivity. Anti-HLA-E monoclonal antibodies (mAbs; e.g., TFL-006 and TFL-007) generated at Terasaki Foundation Laboratory (TFL) differ from commercial HLA-E mAbs in mimicking HLA-la &-lb reactivities of IVIg, which is known to suppress the allo-HLA antibody (Ab) production and the activated CD4+ T cells. Whether HLA-E-mAbs also have the ability to suppress the production of these Abs and activation of T cells like IVIg is evaluated. Indeed, the TFL-mAbs significantly suppressed the allo-HLA-ll Ab production by B cells obtained from a woman alloimmunized postpartum >20 years ago. Similarly, the TFL-mAbs suppressed HLA-I Ab produced by a B-cell hybridoma. In both instances, the suppression was far superior to that by IVIg. Activated T cells were suppressed by both IVIg and the TFL-mAbs dosimetrically. Furthermore, the required concentrations of TFL-mAbs are 150-fold lower than the concentrations of IVIg required for the suppression.

Protective immunity remains intact after antibody removal by means of proteasome inhibition.

BACKGROUND: Proteasome inhibition abrogates donor-specific anti-human leukocyte antigen (HLA) antibody (DSA) in patients posttransplant. However, its effects on protective humoral immunity to vaccine antigens remain unknown. Herein, we report on bortezomib's safety regarding protective immunity in patients who have experienced HLA antibody reduction/removal. METHODS: Thirteen living donor renal transplant patients were treated with bortezomib one to two cycles (1.3 mg/m² × 4 doses) and plasmapheresis in 2008 to remove HLA antibodies posttransplant. Serial measurements of HLA antibody were conducted weekly before, during, and after treatment by means of single antigen bead on Luminex (One Lambda Inc., Canoga Park, CA). Measles and tetanus toxoid IgGs were measured quantitatively by using ELISA (American Research Products Inc., Belmont, MA). RESULTS: All patients treated with bortezomib/plasmapheresis resulted in a primary DSA reduction of more than 50%. In 10 of 13 patients, complete DSA removal (to below 1000 mean fluorescent intensity) occurred. At 1 year posttreatment, antibody intensity remains significantly depressed in the group as a whole. Despite the significant effect on antibody production, tetanus toxoid and measles IgG levels remained unchanged and above the level of protection at 1 year posttreatment. CONCLUSION: These data indicate that proteasome inhibitors plus plasmapheresis results in prolonged reduction of HLA antibodies while leaving protective immunity intact.