An Example of Validation of a Potential Therapeutic Antibody

Background:  Molecule A has been known to express on the epithelial cell membrane of human gastric mucosa. It has also been shown to express on the cell membrane of certain types of carcinomas. Two types (mouse and human) of monoclonal antibodies were generated against the human type molecule A. To explore the therapeutic potential of the human antibody and the target, it was important to determine: 1. expression pattern of the molecule among a variety of normal and tumor tissues; 2. cross-reactivity of the human monoclonal antibody in human tissues. Biotinylated antibodies were prepared. IHC was performed in tissue arrays containing 135 human normal  tissues from 34 sites and 95 tumor tissues from 22 cancer types.

 Procedures

 Results

Picture

 

 

 

IHC result analysis

 

 

Example of a chart showing detailed analysis of the antibody staining patterns across a wide range of normal and tumor tissues. 

 

 

 

IHC with mouse and human antibodies to molecule A in normal tissues

Example of the mouse (MMab, left panel) and human (HMab, right panel) antibody staining in normal tissues. Both antibodies gave strong membrane staining to the glandular epithelial cells in the stomach and ileum. Human antibody also showed cytoplasmic and non-specific staining to other cell types.

 

 

 

IHC with mouse and human antibodies to molecule A in tumor tissues

 

In some types of tumor, the mouse antibody (left panel) showed specific, but heterogeneous membrane staining. The human antibody (right panel) showed membrane and cytoplasmic staining with non-specific background.

 

 

 

IHC with mouse and human antibodies to molecule A in tumor tissues

 

 

In some other types of tumor, the mouse antibody (left panel) showed nuclear staining. The human antibody (right panel) showed predominantly membrane and cytoplasmic staining with non-specific background.

 

 

 

 

 

 

Immunofluorescence staining with mouse antibody to molecule A in a tumor tissue

 

 

 

The mouse antibody dominant membrane staining (red) in the tumor cells.

 

 

 

 

Conclusion

  • The mouse monoclonal antibody detected a general elevation of molecule A in tumors. The staining was highly heterogeneous, i.e. the patterns, positive rates and staining intensity of tumor cells varied greatly among different and/or same types of tumors, or within the same tumors.

  • The human antibody staining was predominantly cytoplasmic and homogeneous. The antibody showed a low specificity to its intended target cells on human tissue sections.

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