Claudia Heuser

Tumor-specific T cell activation by recombinant immunoreceptors : CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule

Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA+ colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3ζ (BW431/26-scFv-Fc-CD3ζ), or FcεRIγ (BW431/26-scFv-Fc-γ). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcεRIγ or CD3ζ signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-γ upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with γ- or ζ-signaling receptors, but enhanced both IFN-γ secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-γ receptor-grafted T cells. Both purified CD4+ and CD8+ T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3ζ signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3ζ) with dual signaling properties. T cells grafted with the combined CD28/CD3ζ signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.

T Cell Activation by Antibody-Like Immunoreceptors: Increase in Affinity of the Single-Chain Fragment Domain above Threshold Does Not Increase T Cell Activation against Antigen-Positive Target Cells but Decreases Selectivity

Chimeric TCRs with an Ab-derived binding domain confer predefined specificity and MHC-independent target binding to T cells for use in adoptive immunotherapy. We investigated the impact of receptor binding affinity on the activation of grafted T cells. A series of anti-ErbB2 single-chain fragment binding domains with a Kd ranging from 3.2 × 10−7 to 1.5 × 10−11 M was linked to CD3ζ-derived immunoreceptors and expressed in human PBL. Solid phase bound ErbB2 protein triggered activation of receptor-grafted T cells in a dose-dependent manner. The activation threshold inversely correlated with the affinity of the receptor binding domain. The maximum level of cellular activation, however, was the same and independent of the binding affinity. Upon binding to ErbB2+ cells, T cells grafted with immunoreceptors carrying a single-chain fragment of Kd < 10−8 M were activated in a similar fashion against cells with different amounts of ErbB2 on the surface. T cells with a low affinity receptor (Kd > 10−8 M), however, were activated exclusively by cells with high amounts of ErbB2. In conclusion, recombinant immunoreceptors of higher affinity do not necessarily induce a more potent activation of T cells than low affinity immunoreceptors, but the higher affinity immunoreceptors exhibit less discrimination between target cells with high or low Ag expression levels.

Tuning tumor-specific T-cell activation: a matter of costimulation?

Abstract The stimulation of a specific antitumor immune response, involving the recruitment of T cells and induction of T-cell effector functions, is an attractive possibility for cancer immunotherapy. In the past few years, advances in our understanding of the mechanisms of T-cell activation and costimulation have provided the basis for strategies to enhance antitumor immunity and break tolerance. These strategies include the equipment of tumor cells with costimulatory molecules such as B7, blockade of inhibitory signals on T cells (e.g. through cytotoxic T-lymphocyte antigen 4) and grafting of T cells with antigen-triggered, recombinant costimulatory receptors. Combining antigen-triggered activation with appropriate costimulatory pathways will lead to novel approaches to improve the efficacy of T-cell-mediated adoptive immunotherapy of malignant diseases.

T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells.

Recombinant TCRs confer specificity to T cells and trigger their activation. Receptors with Ab-derived binding domains have the advantages of MHC-independent Ag recognition and of targeting a variety of chemically different molecules. We explored the impact of the position of a defined epitope within the target molecule on the efficacy of receptor-mediated T cell activation. T cells were grafted with recombinant immunoreceptors that recognize either the membrane distal N or the proximal A3 domain of carcinoembryonic Ag (CEA). Upon binding to isolated, solid-phase immobilized CEA, receptor-mediated T cell activation correlates with the binding efficiency, irrespectively, of the epitope position. Upon binding to CEA expressed on the cell membrane, in contrast, the A3 epitope mediates more efficiently T cell activation than the N epitope, although the N epitope is bound with higher affinity. The CEA N epitope when expressed in a more membrane proximal position, however, activated receptor grafted T cells with higher efficiency than in the distal position. The position of the targeted epitope within the molecule obviously has major impact on the efficacy of T cell activation independently of the binding efficiency of the immunoreceptor.

The Recombinant T Cell Receptor Strategy: Insights into Structure and Function of Recombinant Immunoreceptors on the Way Towards an Optimal Receptor Design for Cellular Immunotherapy

A promising approach in adoptive immunotherapy is based on the induction of a specific cellular anti-tumor response by antigen-specific, cytolytic T cells. Due to difficulties in isolating tumor-specific T cells in sufficient amounts, it was proposed to graft cytolytic T cells with an antigen-specific, recombinant T cell receptor. The antigen binding domain of the receptor consists of a single-chain antibody fragment (scFv) that is derived from a monoclonal antibody and binds to a tumor associated antigen. The intracellular signalling domain is derived from the cytoplasmic part of a membrane bound receptor to induce cellular activation, e.g., the Fc epsilon RI receptor gamma-chain or the CD3 zeta-chain. By use of this type of recombinant receptor, the strategy combines the advantages of MHC-independent, antibody-based antigen binding with efficient T cell activation upon specific binding to the receptor ligand. The modular composition of the receptor, moreover, facilitates modification of both the antigen binding and signalling properties. Accordingly, we and others have generated a panel of recombinant T cell receptors with specificities for malignantly or virally transformed cells. Receptor grafted effector cells were demonstrated to mediate a highly efficient immune response towards antigen expressing target cells. However, little is known about the impact of the recombinant receptor modules on recognition of highly heterologous target antigens and on cellular activation in a complex immunological context. This review summarizes the current knowledge about the generation and function of recombinant immunoreceptors and discusses the limitations and perspectives of the methodology for use in cellular immunotherapy.

Characterization of a chimeric T-cell receptor with specificity for the Hodgkin's lymphoma-associated CD30 antigen.

Recombinant receptors with antibody-like specificity for tumor-associated antigens were shown to direct specifically T cells to target tumor cells. Hodgkin and Reed-Sternberg cells, the malignant cell population in Hodgkins lymphoma, express high amounts of the cell surface antigen CD30. An anti-CD30 T-cell receptor with cellular activation properties is expected to graft T cells with specificity to Hodgkin cells. Here, the authors characterize a chimeric T-cell receptor with an extracellular domain consisting of the single-chain antibody fragment HRS3-scFv with specificity for the CD30 antigen and intracellular domain of the signal transducing part of the Fc-epsilon-I-gamma receptor. The HRS3-scFv was derived from the monoclonal anti-CD30 antibody HRS3 and retained specificity for the CD30 antigen. The recombinant HRS3-scFv-gamma receptor was expressed under control of the RSV-LTR after transfection into MD45 T-cells. The chimeric receptor protein is detected and analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Expression of the chimeric receptor converts MD45 T cells to specificity for CD30+ lymphoma cells. Specific cross-linking of the chimeric receptor with antigen resulted in cytolytic reactivity against CD30+ tumor cells in vitro. The results demonstrate that the chimeric receptor HRS3-scFv-gamma converts T cells to a specific MHC-unrestricted cytolytic response against CD30+ tumor cells offering an alternative strategy in cellular immunotherapy of Hodgkins disease.

CD4+ T Cells Engrafted with a Recombinant Immunoreceptor Efficiently Lyse Target Cells in a MHC Antigen- and Fas-Independent Fashion

T cells engrafted by a recombinant immunoreceptor with predefined Ag specificity can efficiently lyse Ag-positive target cells in a MHC Ag-independent manner. It is yet unresolved how receptor-grafted CD4+ T cells contribute to MHC Ag-independent target cell lysis. To address this issue, we grafted isolated CD8+ and CD4+ T cells from the peripheral blood with recombinant anti-carcinoembryonic Ag and anti-CD30 receptors, respectively. Cytotoxicity analyses revealed that grafted CD4+ T cells exert cytolysis of Ag-positive target cells with an efficiency similar to that of grafted CD8+ T cells. Lysis by receptor-grafted CD4+ T cells is Ag specific and is inhibited by blocking the target Ag or the Ag binding site of the recombinant receptor. Both Fas-sensitive and Fas-resistant target cells are lysed with equal efficiency, and lysis of Fas-sensitive target cells is not blocked by an anti-Fas ligand Ab, indicating that cytolysis by receptor-grafted CD4+ T cells is independent of the Fas pathway. We conclude that cytolysis by CD4+ T cells equipped with a recombinant immunoreceptor is MHC Ag and Fas independent and likely to be mediated by perforin present in receptor-grafted CD4+ T cells.

Isolation of single chain antibody fragments with specificity for cell surface antigens by phage display utilizing internal image anti-idiotypic antibodies.

Recombinant single chain antibody fragments (scFv) with specificity for membrane-bound antigens can be isolated by phage display techniques. The strategy involves selection of recombinant phage antibodies by binding to cells expressing the respective antigen. This results frequently in high nonspecific adherence of phages to cellular membranes. To resolve the problem we have made use of an internal image anti-idiotypic antibody mimicking the membrane-bound CD30 antigen and successfully isolated scFv fragments with specificity for CD30. The cDNA coding for the immunoglobulin heavy and light chain variable regions of the anti-CD30 monoclonal antibody (mAb) HRS3 was expressed by phage display techniques. Recombinant HRS3-scFv phages were efficiently enriched by one cycle of panning on the internal image anti-idiotypic mAb 9G10. The isolated HRS3-scFv clone retained the binding specificity of the parental mAb HRS3 to the internal image anti-idiotypic mAb 9G10 as well as to an anti-idiotypic mAb without the internal image. Furthermore HRS3-scFv reacted with recombinant and cell-bound CD30 antigen, respectively. Binding of scFv fragments to anti-idiotypic mAbs will provide a versatile strategy for the efficient isolation of recombinant antibody fragments.

Anti-CD30-IL-12 antibody-cytokine fusion protein that induces IFN-γ secretion of T cells and NK cell-mediated lysis of Hodgkin's lymphoma-derived tumor cells

Interleukin‐12 (IL‐12) is a disulfide‐linked p40‐p35 heterodimeric cytokine and plays a key role in linking innate cellular immunity to an adaptive Th1 response against pathogens and tumor cells and in counteracting a Th2 immune response. The pathogenesis of Hodgkins disease (HD) is partially attributed to a Th2 dominance associated with functional anergy of T cells that accumulate in the near vicinity to the malignant Hodgkin/Reed‐Sternberg (H/RS) cells. To revert Th2 polarization in the tumor lesion, we generated an anti‐CD30‐IL‐12 antibody‐cytokine fusion protein that binds to CD30 on H/RS cells and is composed of a CD30 binding domain (HRS3‐scFv) linked to p40‐p35 murine single chain IL‐12. The HRS3‐scFv‐hi‐IL‐12 fusion protein is expressed as a 110 kD polypeptide, can be purified by affinity chromatography, and has binding specificities to both the CD30 antigen and the IL‐12 receptor. After binding to CD30+ H/RS cells, the fusion protein stimulates T cells to secrete IFN‐γ, a predominant Th1 cytokine, and induces NK cells to lyse CD30+ cells with high efficiency. These properties make the HRS3‐scFv‐hi‐IL‐12 fusion protein suitable for the specific immunotherapy of Hodgkins lymphoma.

An IL12-IL2-antibody fusion protein targeting Hodgkin's lymphoma cells potentiates activation of NK and T cells for an anti-tumor attack.

Successful immunotherapy of Hodgkins disease is so far hampered by the striking unresponsiveness of lymphoma infiltrating immune cells. To mobilize both adoptive and innate immune cells for an anti-tumor attack we fused the pro-inflammatory cytokines IL2 and IL12 to an anti-CD30 scFv antibody in a dual cytokine fusion protein to accumulate both cytokines at the malignant CD30+ Hodgkin/Reed-Sternberg cells in the lymphoma lesion. The tumor-targeted IL12-IL2 fusion protein was superior in activating resting T cells to amplify and secrete pro-inflammatory cytokines compared to targeted IL2 or IL12 alone. NK cells were also activated by the dual cytokine protein to secrete IFN-γ and to lyse target cells. The tumor-targeted IL12-IL2, when applied by i.v. injection to immune-competent mice with established antigen-positive tumors, accumulated at the tumor site and induced tumor regression. Data demonstrate that simultaneous targeting of two cytokines in a spatial and temporal simultaneous fashion to pre-defined tissues is feasible by a dual-cytokine antibody fusion protein. In the case of IL12 and IL2, this produced superior anti-tumor efficacy implying the strategy to muster a broader immune cell response in the combat against cancer.