Expression Of The Chimeric Anti-cd20 Antibody Mutants, And Its Mechanism

In recent years, antibody therapies for tumor were concerned in clinic. Monoclonal antibodies (mAb) directed against CD20, unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. Anti-CD20 antibodies, which have been approved by American Food and Drug Administration (FDA), include Rituximab (Rituxan(?)), Bexxar^TMand Zevalin^TM, indicating that anti-CD20 antibodies are safe in clinic. Despite clinical success the exact mechanisms of action of various anti-CD20 antibodies remains mostly unclear, which it is necessary to study further. The new anti-CD20 antibody HI47 (a mouse IgG3, k) or its human-mouse chimeric fragments yet have been exploited for our intellectual property. The present study was designed for a mutant clone of anti-CD20 and to identify its biological activities in vitro. The intact human-mouse chimeric anti-CD20 antibody IgG was constructed and expressed stably in CHO cells for ADCC and CMC in vivo.The genes encoding light variable region (VL) and heavy variable region (VH) of anti-CD20 antibody were cloned from HI47 hybridoma cells. The anti-CD20 single chain Fv antibody (ScFv) expression vector pCANTAB 5E cd20ScFv was constructed using phage dispay liberay. One clone (-7) was found different from the others (-2) by sequencing. Ser is substituted by Asn at 26 site in the light chain of -7. The parent or mutant genes VL and VH were cloned into two transitional vectors sspB11 and sspB12, containing constant region of κ and γ-CHl of human antibody, respectively. These two constructed transitional vectors were digested to creat VH+CH1 and VL+CL, respectively. Then the products were cloned into the vector sspA19 to construct a Fab' expression vector pYZFcd20. CD20-7 (S26N in light chain) was expressed with 6.4 mg/L, approximately 3 times of the parent CD20-2. The affinity constant Ka of CD20-7 was about 2×10⁹ M^-1 while that of CD20-2 was about 1 × 10⁹ M^-1. By using an immunofluorescence assay, CD20-7 fragment was a more efficient competitor to HI47 binding than CD20-2: the...