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Identification of a Kb-restricted CTL epitope of beta-galactosidase: potential use in development of immunization
W W Overwijk1, D R Surman, K Tsung
1Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
The use of recombinant and synthetic vaccines in the treatment of cancer has recently been explored using model tumor associated antigens (TAA), many of which do not model the immunological state of affairs in which the TAA is expressed by normal tissues. One potentially useful model Ag is beta-galactosidase (beta-gal). Because the activity of this enzyme is so easily detectable, this gene has been inserted into a large number of recombinant viruses and tumors useful to the cancer vaccinologist. In addition, numerous transgenic mouse colonies that have tissue-specific expression of beta-gal have been developed, enabling the modeling of tolerance to "self" Ags. Since most of these mice have an H-2b background, we generated cytotoxic T lymphocytes (CTL) capable of recognizing beta-gal-expressing tumor cells of C57BL\6 origin and have determined that their restriction element is the K(b) molecule. Using an allele-specific epitope forecast to generate a panel of candidate peptides, we have determined that the K(b)-restricted sequence is DAPIYTNV and corresponds to amino acids 96-103 of the intact beta-gal molecule. A recombinant vaccinia virus (rVV-ES beta-gal96-103) was constructed that encoded the peptide epitope preceded by an endoplasmic reticulum insertion signal sequence. Tumor cells infected with this rVV were recognized by the original CTL that had been used to identify the epitope. Furthermore, splenocytes of mice immunized with a rVV encoding the full-length beta-gal molecule and restimulated with the DAPIYTNV peptide specifically recognized tumor cells expressing beta-gal. The identification of this immunogenic beta-gal sequence enables the modeling of immunization strategies in animal models of malignant disease in which the target antigen is a "self" protein.
Insights
Researchers identified a specific peptide sequence from beta-galactosidase (beta-gal) that can be targeted by cytotoxic T lymphocytes (CTLs). This discovery aids in developing cancer vaccines against self-proteins expressed in tumors.
Area of Science:
- Immunology
- Oncology
- Vaccinology
Background:
- Recombinant and synthetic cancer vaccines are being developed using model tumor-associated antigens (TAAs).
- Beta-galactosidase (beta-gal) is a useful model antigen due to its easy detection and expression in transgenic models, allowing study of self-antigen tolerance.
- Existing models often fail to replicate the immunological state of TAAs expressed in normal tissues.
Purpose of the Study:
- To identify a specific immunogenic peptide from beta-galactosidase (beta-gal) that can be recognized by cytotoxic T lymphocytes (CTLs).
- To establish a model for developing cancer immunization strategies targeting self-proteins.
Main Methods:
- Generated beta-gal-specific CTLs from C57BL/6 mice with an H-2b background.
- Utilized allele-specific epitope forecasting to identify potential K(b)-restricted peptides.
- Constructed a recombinant vaccinia virus (rVV-ES beta-gal96-103) encoding the identified peptide with an endoplasmic reticulum signal sequence.
Main Results:
- Identified the K(b)-restricted immunogenic peptide as DAPIYTNV (amino acids 96-103) from beta-gal.
- Demonstrated that tumor cells infected with the rVV encoding this peptide were recognized by the specific CTLs.
- Showed that splenocytes from mice immunized with full-length beta-gal and restimulated with the peptide specifically recognized beta-gal-expressing tumor cells.
Conclusions:
- The identification of the DAPIYTNV peptide provides a specific target for cancer immunotherapy.
- This finding enables the development of animal models for studying immunization strategies against self-proteins in malignant diseases.
- Facilitates the advancement of cancer vaccines utilizing model antigens like beta-gal.