Renate Engel

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8×A2.1/Kb mouse-derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81–88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81–88), we found that the heterodimeric CD8αβ coreceptor, but not normally expressed homodimeric CD8αα, is required for tetramer binding and functional redirection of TCR-transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 antibody and required more peptide than wild-type (WT) MDM2 TCR+ T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) αβ domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expresion, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Chemotactic migration of human diploid fibroblasts is inhibited by contactinhibin.

Dear Editor: Cellular migration is required for a variety of biological processes such as organ formation or tissue remedeling after tissue injury. In order to function as a controlled process, at least two prerequisites have to be fulfilled: i) migration must be directional and it) migration must be stopped when the original size of a tissue has been achieved. Directional migration or chemotaxis is induced by well defined chemoattractants such as lymphokines (7), collagenous peptides (8), fibronectin (3) and PDGF (9). In addition, only cells directly facing a free space or a wound are allowed to respond to chemoattraetants with migration, while cells surrounded by neighboring cells are migration inhibited (1,2,6). After rebuilding injured tissue by invasion and division of cells, a negative migration--and proliferation signal must be generated. It has long been accepted that cell-cell contacts are required for a stop of migration, although the molecular basis has since not yet been identified. We have shown previously that contactinhihin, a 60-70 kDa plasma membrane glycoprotein is responsible for contact-dependent inhibition of growth (11). Therefore, experiments were undertaken to address the question whether contactinhibin might also be involved in the inhibition of chemotactic migration. Table 1 shows, that the degree of chemotactic migration of human fibroblasts was inversely correlated to the cell density seeded in the upper compartment of the Boyden chamber. At 2 × 105 cells seeded onto the filter, the cell density most commonly used in similar studies, approximately 17% of the cells have migrated after 4 hours. This is in good agreement with experiments where human fibroblasts have been used (9). Chemically transformed mouse fibro-