Towards Adoptive Immunotherapy Using High Affinity T Cell Receptors

Abstract

The hdm2 oncoprotein is frequently overexpressed in a variety of human malignancies, including acute myeloid and lymphoblastic leukemia. Synthetic peptides representative of hdm2 sequences were tested for their binding to A_2.1. HLA-A_2.1. (/K^b)-Tg mice were immunized with A2.1-binding hdm2 peptides in order to induce A2.1-restricted and hdm2-specific CTL. A2.1-restricted CTL lines obtained from both A2.1 and HuCD8 × A2.1/K^b-Tg mice were able to recognize a synthetic 8-mer peptide representative of a N-terminal hdm2 sequence. These CTL were capable of recognizing and lysing Saos-2 cells transfected with the hdm2 gene as opposed to the parental cell line that lacks any detectable hdm2 expression, thereby indicating that the 8-mer peptide is being naturally processed and presented by A2.1. The identity of the naturally presented hdm2 CTL epitope and the synthetic hdm2 8-mer peptide was confirmed by extracting peptides from class I MHC molecules of hdm2-transfectants. Subsequent HPLC-purification allowed the reconstitution of CTL lysis by those natural and synthetic HPLC-fractions which had an identical retention time. CTL specific for the hdm2 8-mer epitope were of sufficiently high avidity to specifically kill A2.1⁺ human leukemia and myeloma targets provided that these cells displayed high level hdm2 protein expression as ascertained by Western blotting. In contrast, a variety of nontransformed human cells, such as PBMC, resting T and B cells, and antigenactivated T lymphocytes, all of them which did not express detectable amounts of hdm2, were not susceptible to lysis by these CTL. The full length a and b chains of high affinity T cell receptors (TCRs) derived from A2.1-restricted and hdm2-specific murine CTL clones have been cloned and sequenced. Hu A2.1-positive dendritic cells pulsed with the leukemia-associated hdm2 self-epitope were able to induce peptide-specific autologous human CTL. These autologous CTL, however, appeared to be of low avidity, thereby indicating that the hdm2 epitope does represent a self-tolerogen for high avidity CTL in vivo.