J. Marcus Muche

Anti‐tumor immune responses and tumor regression induced with mimotopes of a tumor‐associated T cell epitope

Mimotopes provide an alternative to natural T cell epitopes for cancer immune therapy, as they can recruit and stimulate T cell repertoires that deviate from the repertoires engaged with the tumor and exposed to disease‐related immune suppression. Here, mimotopes of a shared tumor‐associated T cell epitope in cutaneous lymphoma were tested for their capacities to induce clinical and immunological responses in cancer patients. The mimotope sequences had been determined by a combinatorial peptide library approach without knowledge of the corresponding natural tumor‐associated antigen. Vaccination with these mimotopes together with helper T cell‐inducing antigens led to complete tumor remission in the two patients tested. After each booster vaccination, enhanced frequencies of mimotope‐specific CD8+ T cells were detected in the peripheral blood of the patients, and the CTL proved to be cytotoxic and tumoricidal when tested in vitro. These data provide a first indication of clinical efficacy of mimotopes in cancer patients.

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma.

Detection of T‐cell clonality by polymerase chain reaction (PCR) and high‐resolution electrophoresis facilitates differentiation of early stages of cutaneous T‐cell lymphoma (CTCL) from benign T‐cell‐rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)‐loaded temperature‐gradient gel electrophoresis (TGGE) to detect clonally expanded T‐cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD‐TGGE detected clonal T‐cell receptor (TCR)‐gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/μL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single‐strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD‐formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/μL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD‐TGGE was investigated by dilution experiments using the well established T‐cell lines Jurkat, Molt‐4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5—10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5—10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1—5% was observed.