Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59

BACKGROUND: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). METHODS: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. RESULTS: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. CONCLUSION: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

Clinical Implications of Pre-Transplantation Panel Reactive Antibody in Renal Transplantation in Koreans / 대한이식학회지

PURPOSE: Panel reactive antibody (PRA) is a screening test for HLA alloimmunization. The purpose of this study is to clarify the clinical implications of pre-transplantation PRA in renal transplantation in Koreans. METHODS: Study subjects included 99 renal transplant recipients whose HLA cross match tests were conducted between Jan. 1994 and Dec. 1995. Their sera were tested for PRA using 50 lymphocyte panels from Koreans by NIH and AHG methods. RESULTS: PRA was positive in 18 (18%) patients by NIH method, and in 19 (19%) patients by AHG method. NIH PRA positivity was higher in the female group (33% vs. 12% in males, P=0.01) while the age of AHG PRA (+) group was higher than the (-) group (37+/-16 vs. 28+/-13, P 45, donor age > 50 and AHG PRA (+) were associated with worse graft survival. In multivariate analysis, donor age > 50 along with AHG PRA (+), or donor age > 50 with recipient age > 45 were significant prognostic factors for graft survival. Recipient age >45, donor age > 50 and AHG PRA (+) were still prognostic of long-term graft fates in cadaveric transplants. CONCLUSION: AHG PRA correlates better with clinical data than NIH PRA and pre-transplant PRA is a significant prognostic factor for long-term graft fates in cadaveric renal recipients in Koreans.

Comparison of NIH-CDC and AHG-CDC methods for the Detection of Panel Reactive Antibodies / 대한임상병리학회지

BACKGROUND: Panel reactive antibody (PRA) test is used for anti-HLA antibody screening and characterization in patients awaiting organ transplantation. Complement-dependent cytotoxicity (CDC) is the most widely used standard procedure and addition of antihuman globulin (AHG) reagent to the basic (NIH) CDC method increases the sensitivity of detection of HLA antibodies. We compared NIH-CDC and AHG-CDC methods for the detection of HLA class I panel reactive antibodies. METHODS: A total of 314 sera from 253 patients were analysed for the detection of HLA class I antibodies by NIH-CDC and AHG-CDC methods using a panel of 50 lymphocytes. PRA% and reaction strength (mean score) were calculated and antibody specificities were identified with r value calculated for antibody specificity. RESULTS: A total of 46 (15%) out of 314 sera were PRA-positive (PRA%> or =10%) by either NIH-CDC (33 sera) or AHG-CDC (43 sera). Concordance of PRA test results between these two methods was 96% (301/314). AHG-CDC was more sensitive in the detection of HLA antibodies compared with NIH-CDC, showing significantly higher PRA% (44% vs 29%, P=0.0001) and reaction strength (mean score 7.3 vs 6.1, P=0.0015) for PRA-positive samples. Among 46 PRA-positive sera, HLA antibody specificities were identified in 21 samples (46%) by NIH-CDC and in 32 samples (70%) by AHG-CDC. AHG-CDC methods frequently detected a wider range of antibody specificities compared with NIH-CDC and provided a more accurate assessment of the antibody specificities. Follow up PRA tests were useful providing information on change of alloimmunization status and antibody specificities in prospective organ transplantation patients. CONCLUSIONS: Compared with NIH-CDC, AHG-CDC method is more sensitive in the detection of panel reactive antibodies and provides a more accurate assessment of the HLA antibody specificities.