HYBRIDOMA Volume 10, Number 1, 1991 Mary Ann Liebert, Inc., Publishers
Recombinant Human-Mouse Chimeric Monoclonal Antibody Specific for Human Adenocarcinoma Associated Antigen MANABU NAKASH1M A, YUSHI NISHIMURA, and TAKESHI WATANABE Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812, Japan
ABSTRACT
recently described a class-switched (IgM to IgGl) human-mouse chimeric human In the present study, a human-mouse chimeric antibody specific for adenocarcinoma-associated antigen YH206 antigen was constructed by fusing murine variable region genes (Vk and Vh) to human constant region genes (yl,K). The murine variable domain genes were isolated from a functional murine hybridoma cell line, YH206, which secreted IgM monoclonal antibody specific for YH206 antigen. The fusion genes of heavy and light chains were introduced into the immunoglobulin nonWe have
antibody.
mouse myeloma cell line X63-Ag8.653 by electroporation. We obtained transformants which secreted class-switched human-mouse chimeric antibodies specific for YH206 antigen. A dot immunobinding assay demonstrated that the classswitched chimeric antibody retained the ability to bind to the YH206 antigen.
producing
INTRODUCTION Monoclonal antibodies specific for human tumor-associated antigens have been the purpose of clinical application, that is, for diagnosis as well as for treatment of cancer1 "5. Although human-human hybridoma technology has been described, only a limited number of human monoclonal antibodies are available. Most of the available monoclonal antibodies are murine antibodies. A major limitation to clinical use of heterologous immunoglobulins is the immune response they elicit The against which may render the antibody ineffective and may harm the patients. constant domain of murine immunoglobulin molecules is probably an important target of antigenic recognition by the human immune system. This portion of the molecule has many physiological functions, except for specific antigen recognition. If the constant portion of murine antibody were to be replaced by the constant portion of the human antibody, the chimeric molecule would retain the ability to bind to antigen and have a much lower antigenicity to humans. Using advanced molecular biology and gene manipulation, it is possible to produce fused proteins from artificially fused genes containing mouse variable domains and human constant domains. The murine monoclonal antibody (IgM, YH206) was raised against human lung adenocarcinoma cell line A-549 and appeared to recognize an adenocarcinoma-associated antigen^. Here, we described the construction of immunoglobulin genes in which the DNA segments encoding the V regions from the heavy and light chains of this murine hybridoma were joined to the DNA segments encoding human y\ and k constant regions. Transfection of expression vectors containing these chimeric immunoglobulin genes into mouse myeloma cells resulted in production of complete
produced for
1
immunoglobulin molecules antigen.
and class switched-chimeric
bind to
IgGl
which had the
ability
to
MATERIALS AND METHODS Cell Lines A murine hybridoma, YH206, which secreted monoclonal antibody ((i,k) specific for human adenocarcinoma-associated antigen, was kindly provided by Dr. Yachi (Sapporo Medical University, Japan). An immunoglobulin non-producing murine myeloma cell line, X63-Ag8.635, a non-producer of immunoblobulin, was obtained from Dr. G. Köhler. These cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum.
DNA isolation and analysis
High molecular weight DNA was isolated from each cell line as described7. Ten micrograms of DNA from each cell line were digested with various restriction enzymes. Digested DNA was separated by electrophoresis on 0.7% agarose gel and was transferred to nitrocellulose filter. Probes were labeled with (32P)-dCTP by nick-translation and were hybridized to filter. The probes used to detect murine light and heavy chains were DNA fragments containing murine JKi.5 region and murine Jh4. respectively. Construction and Screening of
X-phage
libraries
The genes which coded for the light and heavy chains of YH206 were isolated from genomic libraries constructed in bacteriophage X. The libraries were screened by plaque hybridization with nick-translated probes. To clone the gene coding for the light chain, BamHI-digested DNA fractions containing desired fragments were cloned into Charon 28. For the heavy chain gene cloning, DNA was completely digested with EcoRI and cloned into \gt-WES-XB. Transformation of Murine
Myeloma Cells
The chimeric heavy chain gene was inserted into pSV2-gD± plasmid vector^ and the chimeric light chain gene was cloned into pSV?-neo plasmid vector^. Electroporation technique was used to transfect these chimeric immunoglobulin genes into an immunoglobulin non-producing mouse myeloma cell line, X63-Ag8.653, as described1". Stable transformants were screened by resistance to mycophenolic acid (Lilly Co, Ltd) for gpt and G418 (Gibco Laboratories) for neo.
Approximately 5xl06 cells in 20ug of chimeric heavy chain
0.5 ml of phosphate buffered saline (PBS) containing genes were put into an electric chamber (coaxial chamber P/N 402 B.T.X.) and were subjected to an electric field (Amplitude 800V, B.T.X.). After 48 hours of incubation at 37°C, culture medium containing G418 (1 mg/ml) was added for selection of plasmids containing stable transformants. The resultant stable transformants, which appeared about 3 weeks after electroporation, were screened to select clones producing products of the introduced chimeric heavy chain gene by staining the cytoplasmic human heavy chain with FITC-conjugated anti-human y chain antibody.
Immunoblot_analysis The secreted chimeric
immunoglobulins were precipitated from culture supernatants by protein A-sepharose beads. The immunoprecipitates were separated by SDSpolyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Protein bands were transferred to nitrocellulose filters by using a Bio-Rad Trans-Blot Cell apparatus11. To reduce background, filters were treated with Blotto (5% non-fat milk) for 60 min12. Alkaline-phosphatase-conjugated goat anti-human y chain specific and goat anti-human k chain specific IgG were diluted in Blotto and incubated 2
with the filter. After washing with TBS buffer (25 mM Tris. Cl pH7.5, 150 mM NaCl, 0.05% Tween 20), the substrate was added to the filters until a visible sign of reaction
appeared.
RESULTS Isolation of YH206
High
immunoglobulin genes
weight YH206 DNA was digested with various restriction enzymes and analyzed by the method of Southern14. Light chain genes were probed with murine JKi-5 (provided by Dr. T. Honjo, Kyoto University, Japan). A unique light chain gene band was seen at 7.4 kb. This YH206-spccific 7.4 kb BamHI fragment was cloned into Charon 28, and the resultant phage library was screened with the light chain probe. Heavy chain genes were identified by Southern's analysis by using a 1.6 kb fragment containing murine Jh4 (provided by Dr. T. Honjo, Kyoto University, Japan) as a probe. The 2.9 kb YH206-specific EcoRI fragment was ligated into Xgt-WES-XB vector. The library was screened by the heavy chain probe. Partial restriction maps of the isolated YH206 heavy and light chain gene fragments are shown in Figure 1. DNA sequence of these cloned fragments indicated that the heavy chain variable region gene was rearranged to Jh3 and light chain variable region gene was rearranged to JKi (data not shown). molecular
was
Construction of Chimeric
Heavy
Chain Gene
The constant region gene of human yl heavy chain was cloned previously from human plasma cell leukemia cell line ARH77^. The 13 kb human heavy chain constant region gene was prepared from a 21 kb fragment of the cloned yl heavy chain gene, which contained the human heavy chain enhancer region. This fragment was inserted into vector pSV2-neo at EcoRI-BamHI site. The murine heavy chain variable region gene which was used was a 1.3 kb EcoRI-Hindlll fragment. Hindlll site of this fragment was changed to EcoRI site using synthetic EcoRI linker. The resultant 1.3 kb
Recombinant Human-Mouse Chimeric Monoclonal Antibody Specific for Human Adenocarcinoma Associated Antigen MANABU NAKASH1M A, YUSHI NISHIMURA, and TAKESHI WATANABE Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812, Japan
ABSTRACT
recently described a class-switched (IgM to IgGl) human-mouse chimeric human In the present study, a human-mouse chimeric antibody specific for adenocarcinoma-associated antigen YH206 antigen was constructed by fusing murine variable region genes (Vk and Vh) to human constant region genes (yl,K). The murine variable domain genes were isolated from a functional murine hybridoma cell line, YH206, which secreted IgM monoclonal antibody specific for YH206 antigen. The fusion genes of heavy and light chains were introduced into the immunoglobulin nonWe have
antibody.
mouse myeloma cell line X63-Ag8.653 by electroporation. We obtained transformants which secreted class-switched human-mouse chimeric antibodies specific for YH206 antigen. A dot immunobinding assay demonstrated that the classswitched chimeric antibody retained the ability to bind to the YH206 antigen.
producing
INTRODUCTION Monoclonal antibodies specific for human tumor-associated antigens have been the purpose of clinical application, that is, for diagnosis as well as for treatment of cancer1 "5. Although human-human hybridoma technology has been described, only a limited number of human monoclonal antibodies are available. Most of the available monoclonal antibodies are murine antibodies. A major limitation to clinical use of heterologous immunoglobulins is the immune response they elicit The against which may render the antibody ineffective and may harm the patients. constant domain of murine immunoglobulin molecules is probably an important target of antigenic recognition by the human immune system. This portion of the molecule has many physiological functions, except for specific antigen recognition. If the constant portion of murine antibody were to be replaced by the constant portion of the human antibody, the chimeric molecule would retain the ability to bind to antigen and have a much lower antigenicity to humans. Using advanced molecular biology and gene manipulation, it is possible to produce fused proteins from artificially fused genes containing mouse variable domains and human constant domains. The murine monoclonal antibody (IgM, YH206) was raised against human lung adenocarcinoma cell line A-549 and appeared to recognize an adenocarcinoma-associated antigen^. Here, we described the construction of immunoglobulin genes in which the DNA segments encoding the V regions from the heavy and light chains of this murine hybridoma were joined to the DNA segments encoding human y\ and k constant regions. Transfection of expression vectors containing these chimeric immunoglobulin genes into mouse myeloma cells resulted in production of complete
produced for
1
immunoglobulin molecules antigen.
and class switched-chimeric
bind to
IgGl
which had the
ability
to
MATERIALS AND METHODS Cell Lines A murine hybridoma, YH206, which secreted monoclonal antibody ((i,k) specific for human adenocarcinoma-associated antigen, was kindly provided by Dr. Yachi (Sapporo Medical University, Japan). An immunoglobulin non-producing murine myeloma cell line, X63-Ag8.635, a non-producer of immunoblobulin, was obtained from Dr. G. Köhler. These cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum.
DNA isolation and analysis
High molecular weight DNA was isolated from each cell line as described7. Ten micrograms of DNA from each cell line were digested with various restriction enzymes. Digested DNA was separated by electrophoresis on 0.7% agarose gel and was transferred to nitrocellulose filter. Probes were labeled with (32P)-dCTP by nick-translation and were hybridized to filter. The probes used to detect murine light and heavy chains were DNA fragments containing murine JKi.5 region and murine Jh4. respectively. Construction and Screening of
X-phage
libraries
The genes which coded for the light and heavy chains of YH206 were isolated from genomic libraries constructed in bacteriophage X. The libraries were screened by plaque hybridization with nick-translated probes. To clone the gene coding for the light chain, BamHI-digested DNA fractions containing desired fragments were cloned into Charon 28. For the heavy chain gene cloning, DNA was completely digested with EcoRI and cloned into \gt-WES-XB. Transformation of Murine
Myeloma Cells
The chimeric heavy chain gene was inserted into pSV2-gD± plasmid vector^ and the chimeric light chain gene was cloned into pSV?-neo plasmid vector^. Electroporation technique was used to transfect these chimeric immunoglobulin genes into an immunoglobulin non-producing mouse myeloma cell line, X63-Ag8.653, as described1". Stable transformants were screened by resistance to mycophenolic acid (Lilly Co, Ltd) for gpt and G418 (Gibco Laboratories) for neo.
Approximately 5xl06 cells in 20ug of chimeric heavy chain
0.5 ml of phosphate buffered saline (PBS) containing genes were put into an electric chamber (coaxial chamber P/N 402 B.T.X.) and were subjected to an electric field (Amplitude 800V, B.T.X.). After 48 hours of incubation at 37°C, culture medium containing G418 (1 mg/ml) was added for selection of plasmids containing stable transformants. The resultant stable transformants, which appeared about 3 weeks after electroporation, were screened to select clones producing products of the introduced chimeric heavy chain gene by staining the cytoplasmic human heavy chain with FITC-conjugated anti-human y chain antibody.
Immunoblot_analysis The secreted chimeric
immunoglobulins were precipitated from culture supernatants by protein A-sepharose beads. The immunoprecipitates were separated by SDSpolyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Protein bands were transferred to nitrocellulose filters by using a Bio-Rad Trans-Blot Cell apparatus11. To reduce background, filters were treated with Blotto (5% non-fat milk) for 60 min12. Alkaline-phosphatase-conjugated goat anti-human y chain specific and goat anti-human k chain specific IgG were diluted in Blotto and incubated 2
with the filter. After washing with TBS buffer (25 mM Tris. Cl pH7.5, 150 mM NaCl, 0.05% Tween 20), the substrate was added to the filters until a visible sign of reaction
appeared.
RESULTS Isolation of YH206
High
immunoglobulin genes
weight YH206 DNA was digested with various restriction enzymes and analyzed by the method of Southern14. Light chain genes were probed with murine JKi-5 (provided by Dr. T. Honjo, Kyoto University, Japan). A unique light chain gene band was seen at 7.4 kb. This YH206-spccific 7.4 kb BamHI fragment was cloned into Charon 28, and the resultant phage library was screened with the light chain probe. Heavy chain genes were identified by Southern's analysis by using a 1.6 kb fragment containing murine Jh4 (provided by Dr. T. Honjo, Kyoto University, Japan) as a probe. The 2.9 kb YH206-specific EcoRI fragment was ligated into Xgt-WES-XB vector. The library was screened by the heavy chain probe. Partial restriction maps of the isolated YH206 heavy and light chain gene fragments are shown in Figure 1. DNA sequence of these cloned fragments indicated that the heavy chain variable region gene was rearranged to Jh3 and light chain variable region gene was rearranged to JKi (data not shown). molecular
was
Construction of Chimeric
Heavy
Chain Gene
The constant region gene of human yl heavy chain was cloned previously from human plasma cell leukemia cell line ARH77^. The 13 kb human heavy chain constant region gene was prepared from a 21 kb fragment of the cloned yl heavy chain gene, which contained the human heavy chain enhancer region. This fragment was inserted into vector pSV2-neo at EcoRI-BamHI site. The murine heavy chain variable region gene which was used was a 1.3 kb EcoRI-Hindlll fragment. Hindlll site of this fragment was changed to EcoRI site using synthetic EcoRI linker. The resultant 1.3 kb
