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Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity
Marina de Lima Fontes1, Franciny Mara de Lima Neves1, Kelvin Sousa Santos1
1Department of Clinical Analysis, Monoclonal Antibody Laboratory, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, Brazil.
The multiple tolerization subtractive immunization (MTSI) technique successfully generated monoclonal antibodies (mAbs) targeting tumor-specific antigens. This method, using a feasible cyclophosphamide dose, overcomes challenges in producing antibodies against poorly expressed epitopes.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Monoclonal antibodies (mAbs) are crucial for biological research but obtaining those specific to poorly expressed or functionally important epitopes remains challenging.
- The multiple tolerization subtractive immunization (MTSI) technique aims to direct immune responses towards specific epitopes, but cyclophosphamide (CY) treatment can cause high mortality and affect the immune system.
Purpose of the Study:
- To evaluate the efficacy and safety of the MTSI technique in generating tumor-specific monoclonal antibodies.
- To assess the impact of different cyclophosphamide (CY) dosages on mice during the MTSI process.
- To characterize the reactivity of generated antibodies against tumor cells.
Main Methods:
- The MTSI technique was employed using RWPE-1 (tolerogen) and PC-3 (immunogen) cells with varying doses of cyclophosphamide (CY).
- Immunosuppression was monitored via white blood cell counts, and antibody reactivity was assessed.
- Monoclonal antibodies (mAbs) were generated using hybridoma technology and characterized by Western blotting and indirect immunofluorescence.
Main Results:
- A CY dosage of 100 mg/kg body weight was found to be feasible, inducing immunosuppression without excessive mouse mortality.
- Polyclonal antibodies (pAbs) from immunized mice showed increased reactivity towards tumor cells.
- Generated mAbs (2-7A50 and 2-5C11) specifically recognized antigens on tumor cells, not their non-tumor counterparts.
Conclusions:
- The MTSI technique is effective in generating monoclonal antibodies that recognize tumor-specific antigens.
- Optimized cyclophosphamide dosage is critical for the feasibility of the MTSI technique.
- The developed mAbs hold potential for cancer research and diagnostics.
