Expression of Anti-CD4 Human/Murine Chimeric Antibody and Their Killer Tumor Activity

From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were snalyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse ig heavy subgroup Ⅱ(A) and x chain subgroupⅢ, respectively. The VH and VL genes were subcloned into pr1-Expr and Pk Expr respectively, then transfected into XL2-Blue. The VH- Pr1 and VL- pk were trans feeted by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques.We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies.These chimeric antibodies are able to kill tumor cells specifically in vitro.

Tumor radioimmunoimaging of chimeric antibody in nude mice with hepatoma xenograft

IM To study the radioimmunoimaging (RAII) using the human/mouse chimeric Ab to evaluate its targeting activity in animal models.METHODS To chimeric Ab was labeled with 131I. RAII was performed at different intervals after injection of radiolabeled Abs in nude mice with human hepatoma xenograft, and tissue distribution of radioactivity was measured. Comparison was made in the chimeric Ab between the single segment Ab and previous murine mAb against HBxAg.RESULTS The experimental objects developed tumorpositive image after 2 days of radiolabeled Abs injection, and the peak accumulation of radioactivity fell on the 7th day. The tumor/liver ratioactivity of the chimeric Ab, single segment Ab, antiHBx mAb, and the control group was 281±021, 244±016, 460±019, and 096±014, respectively.CONCLUSION The genetic engineering Abs have a considerable targeting activity which can be used as a novel humanized vector in the targeting treatment of liver cancer..

Phase I study of chimeric anti-CD20 monoclonal antibody in Chinese patients with CD20-positive non-Hodgkin＇s lymphoma

Objective： This study was designed to determine the safety, pharmacokinetics and biologic effects of a humanmouse chimeric anti-CD20 monoclonal antibody （SCT400） in Chinese padents with CD20-positive B-cell non- Hodgkin＇s lymphoma （CD20 B-cell NHL）. SCT400 has an identical amino acid sequence as rituximab, with the exception of one amino acid in the CH1 domain of the heavy chain, which is common in Asians. Methods： Fifteen patients with CD20＋ B-cell NHL received dose-escalating SCT400 infusions （250 mg/m2： n=3; 375 mg/m2： n=9; 500 mg/m2： n=3） once weekly for 4 consecutive weeks with a 24-week follow-up period. The data of all patients were collected for pharmacoklnetics and pharmacodynamics analyses. Results： No dose-limiting toxicities were observed. Most drug-related adverse events were grade 1 or 2. Two patients had grade 3 or 4 ncutropenia. Under premedication, the drug-related infusion reaction was mild. A rapid, profound and durable depletion of circulating B cells was observed in all dose groups without significant effects on T cell count, natural killer （NK） cell count or immunoglobulin levels. No patient developed anti- SCT400 antibodies during the course of the study. SCT400 serum half-life （Tin）, maximum concentration （Cmax and area under the curve （AUC） generally increased between the first and fourth infusions （P〈0.05）. At the 375 mg/m2 dose, the T1/2 was 122.5±46.7 h vs. 197.0,75.0 11, respectively, and the Cmax was 200.6±20.2 pg/mL vs. 339.1±71.0 ng/mL, respectively. From 250 mg/m2 to 500 mg/m2, the Cmax and AUC increased significantly in a dose-dependent manner （P〈0.05）. Patients with a high tumor burden had markedly lower serum SCT400 concenmations compared with those without or with a low tumor burden. Of the 9 assessable patients, 1 achieved complete response and 2 achieved partial responses. Conclusions; SCT400 is well-tolerated and has encouraging preliminary efficacy in Chinese patients with CD20＋ B-cell NHL.

The Anti—tumor Effects of an Anti—CD71 Chimeric Antibody in Vitro and Its Distribution in a Tumor Xenograft Model

Objective To investigate the anti-tumor effects in vitro and in vivo distribution of the human/murine chimeric antibody (D2C). Methods The CD71 positive target cells (K562, GEM and SMMC7721) and the effector cells, freshly isolated human PBMC, with the ratio of target cells to effector cells 1:50, were incubated in various dilutions of D2C antibody ( Ab) . Antibody dependent cytotoxicity (AD-CC) was tested by using an LDH-release assay. Instead of effector cells, complement was added to the target cells (GEM, SMMC-7721) with various dilutions of D2C Ab. A method of counting death cells was used in complement dependent cytotoxicity (CDC) assay. Tumor localization and distribution of the chimeric antibody (D2C) were observed by labeling the chimeric Ab with radioiodine(131I) and injecting it into nude mice (Balb/c nu/nu) transplanted with human hepatocellular carcinoma cells (SMMC-7721).Results A significant ADCC was observed with the increased concentration of the D2C Ab. Cytolysis of CD71-positive target cells by the D2C Ab was found in the presence of fresh rabbit complement. Labeled D2C administered by intraperitoneal as well as tumor regional injection, was visualized by SPECT. The distribution of D2C Ab in murine organs and tissues showed that non-specific binding was lower following tumor regional administration than when the antibody was administered by an intraperitoneal injection. The human/murine chimeric antibody (D2C) has in vitro anti-tumor effects and can exert its effects in specific tumor localization. Its distribution and local effects in vivo can be detected by radioimmunoimaging.Conclusion CD71 human/murine chimeric antibody showed marked killing of tumor cells in vitro, and specific recognition and high affinity binding to tumor tissue in vivo

CONSTRUCTION AND EXPRESSION OF A HUMAN-MOUSE CHIMERIC ANTIBODY AGAINST HUMAN BLADDER CANCER

Objective: To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

Anti-Proliferative Effects Induced by Anti-CD4 Human/Murine Chimeric Antibody and Murine Anti-CD4 Monoclonal Antibody

Summary: The effects of chimeric anti CD4 human/murine chimeric antibody and murine anti CD4 monoclonal antibody (McAb) on the proliferation induced by anti CD3 McAb, phytohemagglutinin (PHA), IL 2, and allogeneic cells were studied. The results showed that chimeric anti CD4 antibody and murine anti CD4 McAb could inhibit the proliferation induced by the above inducers and the inhibitory effects were related to the dosage of the antibodies.

Cloning of 3H11 mAb variable region gene and expression of 3H11 human-mouse chimeric light chain

IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method from 3H11 hybridoma cells, using 5′ primers for leader sequences. The 3H11 VL gene was then inserted into humanmouse chimeric light chain expression vector and transfected into murine Sp2/0 myeloma cells.RESULTS DNA sequence analysis indicated that the cloned genes included the whole leader sequences and the mature Ig variable region encoding sequences. After gene transfection, transient expression of chimeric light chain protein was detected.CONCLUSION DNA sequences and transient expression indicated that the cloned gene was functional. This work laid basis for constructing 3H11 humanmouse chimeric antibody in the future..

CD4^+ T Cell Apoptosis Induced by Anti-CD4 Antibodies

To explore the inhibitory effects of anti-CD4 human/murine chimeric antibodies on lymphocyte proliferation, CD4^+ T cell apoptosis induced by anti-CD4 antibodies was examined. Annexin- V -FITC and PI double stain method was employed to qualitatively and quantitatively determined CD4^+ T cell apoptosis induced by anti-CD4 antibodies. Our results showed that anti-CD4 chimeric antibodies could specifically induce CD4^+ T cell apoptosis. The ability of anti-CD4 chimeric antibodies to induce CD4^+ T cell apoptosis was related with the presence of monocytes. It is concluded that the further cross-linking of anti-CD4 antibodies is important for inducing CD4^+ T cell apoptosis.