CLONING AND SEQUENCING OF IMMUNOGLOBULINVARIABLE－REGION GENE OF A MONOCLONALANTIBODY SPECIFIC FOR HUMANHEPATOCARCINOMA

A murine monoclonal antibody HAb27 specific for human hepatocarcinoma has been developed for radioimmunolocalization in animal models. The isotype of this antibody was IgGl, k. In the present study, we used a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable region genes by the polymerase chain reaction. Sequence analysis of the heavy variable region indicated that the VH region was highly homologous to the plasmacytoma cell line MOPC21 gene, and closely related to germline genes of the VHⅢ family. The JH region was encoded by the JH3 gene. For the light chain, the VK segment of the antibody showed the highest homology to the germline VKOXl gene,and the JK region was JK5.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

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.

Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro

OBJECTIVE: To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. METHODS: Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation. RESULTS: Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC. CONCLUSIONS: Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

A study of Hodgkin/Reed-Sternberg cells using single cell polymerase chain reaction

OBJECTIVE: To investigate the characteristics of Hodgkin/Reed-Stemberg (H/R-S) cells found in patients with various types of Hodgkin's disease (HD). METHODS: H/R-S cells were micropicked from frozen sections of tissues affected by HD. The DNA from these cells was amplified by polymerase chain reaction (PCR) using immunoglobulin heavy chain gene FR III a/JH primers and light chain gene family-specific primers. RESULTS: A total of 52/135 (35.8%) isolated cells showed the specific products in the reactions. IgH and V kappa 4 rearrangements were repeatedly found in many cells from a lymphocyte predominance type sample; repeated V kappa 4 and individual IgH/V kappa 2,4 rearrangements and individual IgH, V lambda 3/ V kappa 4 rearrangements were found in two different cases of the nodular sclerosis type; repeated IgH/ V lambda 3 and individual V lambda 2,4 rearrangements, repeated V kappa 2,4 rearrangements, repeated V kappa 4 and individual IgH/ V kappa 3 rearrangements, repeated IgH and individual V kappa 3/ V lambda 4 rearrangements were detected in 3 cases of the mixed cellularity type. Repeated and individual IgH rearrangements were found in other 2 cases. CONCLUSION: The H/R-S cells isolated from the lymphocyte predominance subtypes of HD have IgH and V lambda 4 gene rearrangements. This suggests that the lymphocyte predominance type is a proliferation of neoplastic B cells. The cells isolated from the mixed cellularity and nodular sclerosis types derive from B lineage cells at various stages of differentiation because of the presence of their IgH, kappa and/or lambda gene rearrangements. To our knowledge, this is the first time that the lambda gene rearrangement was detected in H/R-S cells.