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Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Published on: January 8, 2014
Preparation and characterization of a chimeric CD19 monoclonal antibody
H Zola1, P J MacArdle, T Bradford
1Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, South Australia.
Insights
A new chimeric antibody targeting CD19, FMC63, retains specificity for B lymphocytes but exhibits human IgG1 properties. This engineered antibody can fix human complement, offering potential for B-cell lineage immunotherapy.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- CD19 antibodies target B lymphocyte lineage cells, making them candidates for attacking B-cell leukaemias and lymphomas.
- FMC63 is a mouse monoclonal antibody recognizing CD19, a B-lineage specific antigen.
Purpose of the Study:
- To engineer a mouse-human chimeric antibody based on FMC63 for potential immunotherapy.
- To evaluate the properties and specificity of the chimeric antibody, including its interaction with human complement and cytotoxic potential.
Main Methods:
- Gene engineering to create a chimeric antibody with mouse FMC63 variable regions and human IgG1 constant regions.
- Transfection into a mouse myeloma cell line for antibody secretion.
- Purification and biotinylation of the chimeric antibody for specificity determination.
- Assessment of complement fixation and antibody-dependent cellular cytotoxicity (ADCC).
Main Results:
- The chimeric antibody demonstrated a reaction profile consistent with the original FMC63 antibody.
- The engineered antibody exhibited human IgG1 characteristics, including the ability to fix human complement.
- The chimeric antibody was not cytotoxic in vitro, even with complement or effector cells for ADCC.
Conclusions:
- The chimeric FMC63 antibody retains B-cell lineage specificity and gains human IgG1 effector functions like complement fixation.
- The lack of in vitro cytotoxicity warrants further investigation into potential reasons and strategies for therapeutic application.
- The engineered antibody holds promise for B-cell targeted immunotherapies, potentially overcoming limitations of the original mouse antibody.
Abstract:
FMC63 is an IgG2a mouse monoclonal antibody belonging to the CD19 cluster. CD19 antibodies react with a 95kDa protein expressed by cells of the B lymphocyte lineage, from pre-B cells to mature B lymphocytes. CD19 antibodies have been suggested as candidates for immunological attack on leukaemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind, we have produced a mouse-human chimera in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed.
