[High level expression of chimeric antibody fragment F(ab')2 directed against CD20 in Escherichia coli].

The use of tumor antigen specific antibody for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. In previous work, the human-mouse chimeric antibody fragment Fab' directed against CD20 was constructed from the new anti-CD20 antibody HI47 (a mouse IgG3, K). The chimeric antibody fragment Fab' could reduce its antigenicity, but the yield, quality and affinity of chimeric antibody fragment Fab' restrict its use. To improve affinity of chimeric antibody fragment Fab', a new phasmid pYZcpp3, which expresses chimeric antibody fragment F(ab')2, was constructed by adding a sequence encoding a small peptide, (CPP)3, to C-terminus of heavy chain constant region of chimeric antibody fragment Fab'. Using the pYZcpp3 to transform E. coli. 16c9, the genetically engineered bacteria 10916# was obtained. 10916# can secret the soluble chimeric antibody fragment Fab' and F(ab')2 into periplasmic. The yield was up to 360 mg/L with the percent of F(ab')2 up to 45% in 19L fermentor by the high density fermentation technology. Without denaturation and renaturation, the F(ab')2 has possessed the native three-dimensional structure. The purity of F(ab')2 was more than 90% after the purification of protein G affinity chromatography and S200 size exclusion chromatography. The F(ab')2 could distinguish and bind to Raji cells (CD20+) by FACS. F(ab')2 could inhibit the proliferation of Raji cells in vitro by MTT, IC50 was 22.8 microg/mL. HI47 and its chimeric fragments F(ab')2 induced a significant level of apoptosis (23.5%, 20.8%, respectively), independent of any cross-linking agents, in Raji cells after 24 h incubation. The chimeric antibody fragment F(ab')2 directed against CD20 is possible to apply to tumor therapy in clinic in the future.

[Apoptosis of human B cell lymphoma Raji cells induced by monovalent anti-CD20 antibody].

BACKGROUND & OBJECTIVE: The anti-CD20 antibodies and fragments have been applied for treatment of non-Hodgkin's lymphomas (NHL) in clinic. The new anti-CD20 antibodies and their fragments (unmodified or radiolabeled) yet have been exploited for those patients with incomplete response to rituximab. The chimeric antibody fragments Fab and F(ab) '(2) derived from HI(47)(a mouse anti-CD20 antibody) have been constructed. The present study was designed to determine the effect of HI(47) and chimeric anti-CD20 antibody fragments on growth inhibition and apoptosis of lymphoma cells. METHODS: The binding of anti-CD20 antibodies to CD20 positive human B cell lymphoma Raji cells was examined using immunofluorescence assay. MTT method was used to evaluate the effect of chimeric antibody fragments on Raji cells growth. Annexin V staining and DNA ladder were used to examine the apoptosis of Raji cells induced by chimeric antibody fragments. RESULTS: HI(47) and its chimeric antibody fragments were capable of binding to CD20 positive Raji cells with the binding rate of above 90%. HI(47) did not compete with rituximab in binding with Raji cells. The growth of Raji cells was inhibited by HI(47) and its fragments with the inhibition rates of (57+/-1.5)%, (65.2+/-2.5)%,and (77.2+/-3.2)%, respectively, at the concentration of 100 microg/ml. The monovalent antibody fragment Fab (20 microg/ml) induced the apoptosis of Raji cells with early apoptosis rate of 17%. CONCLUSION: The chimeric anti-CD20 antibody fragments derived from HI(47) have inhibitory effect on Raji cells and can induce apoptosis of Raji cells.

[One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

Monoclonal antibodies (mAb) directed against CD20, either 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. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.

[Expression of chimeric anti-CD3 IgG antibody in mammalian cells and analysis of its biological activity].

The anti-CD3 antibody can improve success rate of organs transplant. HIT3a, a mouse anti-CD3 antibody, was chimerized by using gene engineering methods to decrease its immunogenity. The anti-CD3 genes, heavy chain and light chain, were cloned using PCR from the vector pCANTAB 5E containing anti-CD3 scFv gene fragment, and two PCR fragments were recombined into the expression vector pKN100 with human antibody light constant domain and pG1D105 with human antibody heavy constant domain, respectively. The two vectors were co-transfected into CHO cells using liposome. The anti-CD3 antibody was detected by ELISA and Western blot assay in supernatant of transfected CHO cells culture. The primary results of competitive assays by FACS showed that anti-CD3 antibody could partially block the sites through which parent antibody (HIT3a) bind to CD3+ Jurkat cells. The result of 3H-TdR incorporation showed that the chimeric anti-CD3 antibody could stimulated proliferation of peripheral blood mononuclear cells (PBMC) as the parent antibody. In this thesis, the results of some experiments indicated that the chimeric anti-CD3 antibody expressed in CHO cells was an antibody with native biological activity, and it is possible to apply to in clinic in the future.