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Copyright of Der Onkologe is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A majority of cancer deaths are because of an uncontrolled relapse of the disease despite initial remission after therapy, asking for strategies to control tumour cells in the long term. Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed promising success in primary tumour elimination; the capacity of such engineered T cells to establish enduring tumour protection is currently a matter of discussion, in particular as most targeted 'tumour-associated antigens' are self-antigens. To address the issue in a clinically relevant model that closely mimics the human situation, we recorded rejection of carcinoembryonic antigen (CEA)-positive pancreatic tumours in the CEA transgenic mouse that expressed CEA as self-antigen in healthy cells of the gastrointestinal tract. Adoptive therapy with CD8+ T cells, which were redirected by a CEA-specific, low-affinity CAR with CD3ζ endodomain, eliminated CEA+ tumours in a primary response; cured mice produced an efficient recall response in the long term towards CEA+ tumour cells upon rechallenge. Secondary tumour rejection was CEA specific, mediated by engineered T cells and did not require host T cells. No toxicity towards healthy tissues with CEA expression was recorded. Data indicate that adoptive therapy with engineered T cells can establish self-antigen-specific tumour protection in the long term without autoimmunity. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy of cancer using chimeric antigen receptor (CAR)-engineered T cells with redirected specificity showed efficacy in recent trials. In preclinical models, 'second-generation' CARs with CD28 costimulatory domain in addition to CD3ζ performed superior in redirecting T-cell effector functions and survival. Whereas CD28 costimulation sustains physiological T-cell receptor (TCR)-CD3 activation of naïve T cells, the impact of CD28 cosignalling on the threshold of CAR-mediated activation of pre-stimulated T cells without B7-CD28 recruitment remained unclear. Using CARs of different binding affinities, but same epitope specificity, we demonstrate that CD28 cosignalling neither lowered the antigen threshold nor the binding affinity for redirected T-cell activation. 'Affinity ceiling' above which increase in affinity does not increase T-cell activation was not altered. Accordingly, redirected tumor cell killing depended on the binding affinity but was likewise effective for CD3ζ and CD28-CD3ζ CARs. In contrast to CD3ζ, CD28-CD3ζ CAR-driven activation was not increased further by CD28-B7 engagement. However, CD28 cosignalling, which is required for interleukin-2 induction could not be replaced by high-affinity CD3ζ CAR binding or high-density antigen engagement. We conclude that CD28 CAR cosignalling does not alter the activation threshold but redirects T-cell effector functions. [ABSTRACT FROM AUTHOR]

Copyright of Der Pathologe is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Chimeric antigen receptors (CARs, immunoreceptors) are frequently used to redirect T cells with pre-defined specificity, in particular towards tumour cells for use in adoptive immunotherapy of malignant diseases. Specific targeting is mediated by an extracellularly located antibody-derived binding domain, which is joined to the transmembrane and intracellular CD3ζ moiety for T-cell activation. Stable CAR expression in T cells, however, requires a spacer domain interposed between the binding and the transmembrane domain and which is commonly the constant IgG1 Fc domain. We here revealed that CARs with Fc spacer domain bind to IgG Fc gamma receptors (FcγRs), thereby unintentionally activating innate immune cells, including monocytes and natural killer (NK) cells, which consequently secrete high amounts of pro-inflammatory cytokines. Engineered T cells, on the other hand, are likewise activated by FcγR binding resulting in cytokine secretion and lysis of monocytes and NK cells independently of the redirected specificity. To reduce FcγR binding, we modified the spacer domain without affecting CAR expression and antigen binding. Engineered with the modified CAR, T cells are not activated in presence of FcγR+ cells, thereby minimizing the risk of off-target activation while preserving their redirected targeting specificity. [ABSTRACT FROM AUTHOR]

Recent insight into the balance of self-tolerance and auto-aggression has raised interest in using human regulatory T (Treg) cells for adoptive immunotherapy of unlimited autoimmune diseases including type-1 diabetes, rhematoid arthritis and multiple sclerosis. The therapeutic use of Treg cells, however, is so far hampered by the inefficiency of current protocols in making them accessible for genetic manipulations. We report here that TCR/CD3 stimulation that is accompanied by extensive CD28 costimulation makes human Treg cells susceptible to retroviral gene transfer ex vivo while preserving their properties in vitro and in vivo. To show the power of genetic manipulation of human Treg cells, we engineered ‘designer Treg cells’ by retroviral expression of a chimeric immunoreceptor with defined specificity, which activates Treg cells in a ligand-dependent manner to proliferate, to secrete high amounts of interleukin-10 and to repress an ongoing cytolytic T-cell response in vivo. The procedure in genetically modifying human Treg cells ex vivo will open a panel of applications for their use in the adoptive therapy of deregulated immune responses. [ABSTRACT FROM AUTHOR]

Human T lymphocytes can be redirected with a new defined specificity by expression of a chimeric T-cell receptor (immunoreceptor) for the use in adoptive immunotherapy of cancer. Whereas standard procedures use retroviral gene transduction to constitutively express immunoreceptors in T cells, we here explored for the first time mRNA electroporation to achieve transient immunoreceptor expression, and thereby minimizing the risk of persistence of potential autoaggression. CD4+ and CD8+ T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Immunoreceptor-transfected T cells were specifically activated upon coincubation with ErbB2+ and CEA+ tumor cells, respectively, resulting in secretion of interferon-γ (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNFα). Furthermore, immunoreceptor-transfected CD8+ T cells specifically lysed ErbB2+ and CEA+ tumor cells, respectively. The RNA-transfected T cells retained their cytotoxic function after 2 days of activation and exhibited cytolytic activities like retrovirally transduced T cells. RNA electroporation of T cells thereby provides a versatile tool for transient immunoreceptor expression, which may be of advantage in avoiding the persistence of unintended autoaggression.Gene Therapy (2009) 16, 596–604; doi:10.1038/gt.2008.189; published online 22 January 2009 [ABSTRACT FROM AUTHOR]

MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expression is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours. [ABSTRACT FROM AUTHOR]

Recombinant immunoreceptors are modularily composed of extracellular antigen binding and intracellular signalling domains that are preferentially derived from CD3? or Fc?RI?. The impact of the signalling domain on the stability of immuoreceptor expression and function is not completely understood. To address this issue, we generated and expressed a panel of recombinant ?- and ?-chain immunoreceptors, respectively, in human peripheral blood T cells. The expression level of ?-chain immunoreceptors in human T cells is significantly lower than those of the homologous ?-chain receptors. Low ?-chain receptor expression in peripheral T cells is because of the intracellular signalling domain and independent of the Fc?RI? or CD3? transmembrane region. Expression of both receptors decreases upon prolonged cultivation. Shortly after receptor engraftment, target cell lysis and induction of IFN-? secretion are mediated with similar efficiency by ?- and ?-chain immunoreceptors. Upon prolonged propagation, however, T-cell activation mediated by ?-chain immunoreceptors is more efficient than by ?-chain receptors, indicating that the initial high expression level of ?-chain immunoreceptors compensates its lower activation capacity. Consequently, ?-chain immunoreceptors exhibit a higher threshold value for specific activation and are more pronouncedly inhibited by soluble ligand antigen compared to the homologous ?-chain receptor. These findings have substantial consequences for the design of recombinant immunoreceptors for use in adoptive immunotherapy.Gene Therapy (2003) 10, 1408-1419. doi:10.1038/sj.gt.3302023 [ABSTRACT FROM AUTHOR]

In early stages of cutaneous T cell lymphoma (Sézary syndrome) both CD4[SUP+]CD7[SUP-] and CD4[SUP+]CD7[SUP+] T cells clonally expand whereas in late stages of the disease CD7[SUP-] cells are predominant in number, giving rise to the question whether CD7[SUP-]T cells have a survival advantage in the skin. Galectin-1, a cell-bound lectin, was recently reported to trigger apoptosis in activated CD7[SUP+] T cells. Here, we demonstrate that in contrast to activated CD7[SUP+] T cells, quiescent and activated CD69[SUP+]CD7[SUP-] T cells from healthy donors and from Sézary patients are resistant to galectin-1-mediated cell death. CD7[SUP-] T cells are apoptosis-resistant even during coculture with IFN-γ-stimulated endothelial cells that constitutively express galectin-1 in high amounts. These data imply that resistance of CD7[SUP-] T cells to galectin-1-induced apoptosis may contribute to the accumulation of CD7[SUP-] Sézary T cells during progression of the disease. [ABSTRACT FROM AUTHOR]

T cells can be directed to antigen-specific, MHC-independent target ceil lysis by grafting with a recombinant receptor with antibody-like specificity. Here, we asked whether T cells from the peripheral blood of a patient with cutaneous T cell lymphoma can be recruited for an immune response against autologous tumor cells. Lymphoma cells with a CD3[sup -] CD4[sup +] CD30[sup +] phenotype and clonal TCR-Vβ7 rearrangement were isolated from a cutaneous lesion. The lymphoma lesion additionally harbored CD3[sup +] CD25[sup +] activated normal T cells despite ongoing tumor progression. Peripheral blood-derived T cells from the lymphoma patient were retrovirally engrafted with a recombinant anti-CD30scFv-γ receptor. Upon cocultivation with autologous CD30[sup +] lymphoma cells, grafted T cells increase IFN-γ secretion and lyse specifically lymphoma cells with high efficiency, even at an effector to target cell ratio of as low as 1:20. Our data demonstrate that the recombinant anti-CD30-γ receptor overcomes T cell tolerance for tumor cells and directs T cells specifically against autologous lymphoma cells. [ABSTRACT FROM AUTHOR]

The CD7[sup -] subset of CD4[sup +] T cells reflects a stable differentiation state of post-thymic helper T cells with CD45R0[sup +]CD45RA[sup -] 'memory' phenotype. Here we report that CD4[sup +]CD7[sup -] T cells are prone to increased spontaneous apoptosis in vitro compared to CD4[sup +]CD7[sup +] T cells. Spontaneous apoptosis is prevented by IL-15, but not by IL-2. Moreover, IL-15 increases Bcl-2 and decreases CD95/Fas expression of CD7[sup -], but not of CD7[sup +] T cells. Because IL-15 is physiologically not secreted but expressed in a membranebound form, we cocultured T cells with TNF-α stimulated fibroblasts that expose membrane IL-15. TNF-α stimulated fibroblasts rescue CD4[sup +]CD7[sup -] Tcells from apoptosis whereas unstimulated fibroblasts do not. Rescue from apoptosis requires cell-cell contact and is abolished by addition of neutralizing antibodies to IL-15. We conclude that membrane IL-15 prevents accelerated apoptosis of CD4[sup +]CD7[sup -] T cells. This mechanism may contribute to accumulation of CD7[sup -] T cells in chronic inflammatory skin lesions. [ABSTRACT FROM AUTHOR]

T cells can be endowed with antigen specificity by grafting with a chimeric receptor consisting of an extracellular antigen binding moiety (scFv) derived from an antibody and an intracellular signaling domain. Conflicting data exist on the impact of an extracellular spacer domain between the antigen binding and the signaling domain with respect to cellular activation. Here, we recorded conjugate formation and antigen-driven cellular activation of T cells grafted with receptor molecules that contain the same antigen binding site (antiCD30 HRS3-scFv) and signaling domain (Fc∈RI γ-chain), however, with and without an IgG1 CH2CH3 (Fc) spacer domain between the scFv and transmembrane moiety. Receptors of both configurations mediate equally efficient conjugate formation between receptor grafted T cells and antigen-positive target cells. Specific signaling by the spacer containing receptor, however, is blocked by five- to 10-fold lower concentrations of soluble antigen than by the spacerless receptor indicating a higher avidity of the spacer containing receptor to soluble antigen. In contrast, cellular activation upon binding to antigen-positive cells is mediated more efficiently by the spacer-less receptor. This demonstrates that the extracellular spacer domain impairs antigen-dependent cellular activation by the chimeric immune receptor, but not intercellular conjugate formation. [ABSTRACT FROM AUTHOR]

Chimeric T cell receptors with specificity for tumor-associated antigens are successfully used to target T cells to tumor cells. The efficacy of this approach, however, is reduced by soluble antigen that is frequently present in high serum concentrations. To overcome this situation, we constructed an anti-CEA chimeric receptor whose extracellular moiety is composed of a humanized single chain antibody fragment (scFv) derived from the anti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG. The intracellular moiety consists of the γ-signaling chain of the human FcεRI receptor constituting a com- pletely humanized chimeric receptor. After transfection, the humBW431/26 scFv-CH2CH3-γ receptor is expressed as a homodimer on the surface of MD45 T cells. Co-incubation with CEA+ tumor cells specifically activates grafted MD45 T cells indicated by IL-2 secretion and cytolytic activity against CEA+ tumor cells. Notably, the efficacy of receptor-mediated activation is not affected by soluble CEA up to 25 μg/ml demonstrating the usefulness of this chimeric receptor for specific cellular activation by membrane-bound CEA even in the presence of high concentrations of CEA, as found in patients during progression of the disease. [ABSTRACT FROM AUTHOR]