4. Cancer is a disease characterized by uncontrolled cellular growth and accumul

Question

4. Cancer is a disease characterized by uncontrolled cellular growth and accumulation of mutations in the tumor cell. This can result in specific immunogenic tumor-associated antigens (TAA, or neoantigens) in the tumor cell that generate an immune response very much like non-self antigens. A. (16pts.) A patient is suffering from late-stage melanoma (skin cancer). Briefly describe an experimental approach that identifies specific TAA (neoantigen) proteins, peptides or epitopes in this patient's tumor cells. B. (4pts.) Using the TAA you identified (in A, above), provide an idea about how could you use the TAA to develop a personalized immune therapy against the patient's tumor cells?

Explanation / Answer

EXPERIMENTAL APPROACH TO IDENTIFY TAA:

Several approaches are currently available for the identification of TAAs in cancer. One of the approaches is the utilization of serum antibodies from cancer patients to immunoscreen cDNA expression library to identify TAAs in cancer, and some of these identified TAAs may have potential diagnostic values in cancer diagnosis. Another approach involving the use of a proteome-based methodology, which is generally named as immunoproteomics.

The methods which we have used in the identification of putative TAAs has involved initially examining the sera from cancer patient using extracts of tissue culture cells as source of antigens in Western blotting and by indirect immunofluorescence on whole cells. With these two techniques, we identify sera which have high-titer fluorescent staining or strong signals to cell extracts on Western blotting and subsequently use the serum antibodies either in isolating cDNA clones from cDNA expression libraries or in immunoproteomics to identify tumor-associated proteins. Using the approach of immunoscreening cDNA expression libraries, TAAs have been identified. Several antigens were identified using a methodology called SEREX (serological analysis of recombination cDNA expression libraries). Immunoscreening of cDNA libraries with serum antibodies for identifications of autoantigens is a well-established method and has been used not only to identify TAAs but also antigens in autoimmune diseases. This methodology was the basis of the methods described in SEREX with the difference that cDNA expression libraries constructed from autologous patient tumor were used as substrate in immunoscreening. Subsequent reports using the SEREX technique have shown that the TAAs identified are no different from standard methods using cDNA expression libraries from cell lines derived from different sources, so that there did not appear to be any advantage to using cDNA libraries from autologous patients.

A brief description of the immunoproteomic approach we have used to identify and characterize TAAs is shown in Fig. 1. Briefly, the sera from the cancer patient were initially examined using extracts of tissue culture cells as the source of antigens in western blot and by indirect immunofluorescence (IIF) on whole cells. With these two techniques, we identified sera that have high-titer fluorescent staining or strong signals to cell extracts on western blot and subsequently used the antibodies in these sera as probes in a proteomic approach to isolate potential TAAs. Cell extract of cultured cancer cells was applied onto the first dimension isoelectrofocusing gel (1D-IEF), and subsequently loaded onto the second-dimension gel (2D-SDS-PAGE). The protein was transferred to the nitrocellulose membrane or visualized by Coomassie blue staining (or silver staining). After immunoblotting with cancer sera and normal human sera (as controls), a number of protein spots of interest were excised from the 2D gels, digested by trypsin, and subsequently analyzed by mass spectrometry (MS).

Fig. 1: immunoproteomic approach we have used to identify and characterize TAAs

Reference:

Zhu Q, Liu M, Dai L, Ye H, Han S, Zhang J-Y. Using immunoproteomics to identify tumor-associated antigens (TAAs) as biomarkers in cancer immunodiagnosis. Autoimmunity reviews. 2013;12(12):1123-1128. doi:10.1016/j.autrev.2013.06.015.

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