Mutation Analysis of IgVH Gene in B-Cell Chronic Lymphocytic Leukemia

Summary: The variable heavy chain region (VH) genes of 3 untreated patients with B cell chronic lymphocytic leukemia (B CLL) were cloned and analyzed. The VH family used was VH3 11, VH3 72 and VH3 33. More than 2 % difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post GC cells.

Construction,Expression and In Vitro Biological Behaviors of Ig scFv Fragment in Patients with Chronic B Cell Leukemia

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia （B-CLL） and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells （PBMC） was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS- PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD （1 kD= 0. 9921 ku）, which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coil, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id- HSP complex vaccine for B-CLL.

Preparation and Characterization for Bispecific Antibodies of Anti-CD3×Anti-Idiotype to BCellLym phocytic Leukem ia

Bispecific antibodies (BsAbs) of anti CD3×anti idiotype (Id) to B cell lymphocytic leukemia (CLL) were prepared by chemical conjugation and direct hybridization technique of hybridoma and hybridoma without screening markers. The specificity of BsAbs from culture supernatants or ascites was assayed by indirect ELISA and indirect immunoflurescence (IF). The results showed that BsAbs could specifically react with homologous serum IgM from patients with B CLL and cells carrying CD3 marker respectively. Cell combination test and LDH assay demonstrated that BsAb significantly increased the conjugate formation between lymphocyte activated kill (LAK) cells and Daudi cells, and enhanced the cytotoxic activity of LAK cells against Daudi cells.

ANALYSIS ON EPITOPES OF IGM WITH MONOCLONAL ANTI-ISOTYPIC AND ANTI-IDIOTYPIC ANTIBODIES AGAINST IgM FROM B-CLL

A double antibodies additivity ELISA test was employed to identify the epltopes which can be recognized by monoclonal antibodies (McAbs) against IgM from B chronic lymphocyte leukemia (B-CLL). The computer grouping programme analysis showed that 4 and- isotypic MaAbs could be divided into two groups and 10 anti- idiotype McAbs could be divided into four groups. The result was consistent with that of the indirect sandwich ELISA and inhibition ELISA test. It suggested that there were at least 6 distinct IgM epitopes which can react specifically with 14 McAbs. Our study indicated that the combination of the additivity ELISA test and the computer grouping programme analysis is of help in studying the relationship of the structure and function of antigen.

A risk score system for stratifying the risk of relapse in B cell acute lymphocytic leukemia patients after allogenic stem cell transplantation

Background:For patients with B cell acute lymphocytic leukemia(B-ALL)who underwent allogeneic stem cell transplantation(allo-SCT),many variables have been demonstrated to be associated with leukemia relapse.In this study,we attempted to establish a risk score system to predict transplant outcomes more precisely in patients with B-ALL after allo-SCT.Methods:A total of 477 patients with B-ALL who underwent allo-SCT at Peking University People’s Hospital from December 2010 to December 2015 were enrolled in this retrospective study.We aimed to evaluate the factors associated with transplant outcomes after allo-SCT,and establish a risk score to identify patients with different probabilities of relapse.The univariate and multivariate analyses were performed with the Cox proportional hazards model with time-dependent variables.Results:All patients achieved neutrophil engraftment,and 95.4%of patients achieved platelet engraftment.The 5-year cumulative incidence of relapse(CIR),overall survival(OS),leukemia-free survival(LFS),and non-relapse mortality were 20.7%,70.4%,65.6%,and 13.9%,respectively.Multivariate analysis showed that patients with positive post-transplantation minimal residual disease(MRD),transplanted beyond the first complete remission(≥CR2),and without chronic graft-versus-host disease(cGVHD)had higher CIR(P<0.001,P=0.004,and P<0.001,respectively)and worse LFS(P<0.001,P=0.017,and P<0.001,respectively),and OS(P<0.001,P=0.009,and P<0.001,respectively)than patients without MRD after transplantation,transplanted in CR1,and with cGVHD.A risk score for predicting relapse was formulated with the three above variables.The 5-year relapse rates were 6.3%,16.6%,55.9%,and 81.8%for patients with scores of 0,1,2,and 3(P<0.001),respectively,while the 5-year LFS and OS values decreased with increasing risk score.Conclusion:This new risk score system might stratify patients with different risks of relapse,which could guide treatment.