Communication patterns in families affected by parental cancer from the healthy parents' perspective-process evaluation of the complex intervention Family-SCOUT.

PURPOSE: Within families affected by parental cancer, open communication impacts the well-being of parents and their children; however, limited research exists on communication patterns in these families. This sub-study addresses this through the Family-SCOUT study, a multicenter, prospective, interventional, and non-randomized investigation with intervention (IG) and control group (CG). The purpose of this sub-study was to identify and compare the differences in communication patterns between the IG and CG as part of the process evaluation. The research question was addressed in both groups: What communication patterns do healthy parents perceive within their families? METHODS: Using a qualitative approach, the study involved interviewing healthy parents as surrogates for their families. The interviews were audio-recorded, transcribed, and coded using a template analysis. The resulting data were analyzed at the group level. RESULTS: Twenty-three interviews were conducted in the IG and 27 interviews in the CG. The analysis of themes centered on communication patterns as seen in the family structure. Both groups exhibited instances of open communication about fears and wishes as well as the use of child-friendly language when discussing cancer. Notable differences were observed: challenges in open communication with children were sorely reported in CG interviews, and "the illness is discussed when necessary" was sorely described in IG interviews. CONCLUSION: This study underscores the need to address and encourage open communication within families with parental cancer.

Effectiveness of a comprehensive support program for families with parental cancer (Family-SCOUT): results of a multicenter non-randomized controlled trial.

BACKGROUND: Cancer patients with minor children but also their families suffer from significant psychological distress and comorbidity. Protective factors predicting successful coping are well known. Corresponding systematic interventions are rare and limited by access barriers. We developed a comprehensive family-centered intervention for cancer patients with at least one dependent minor. PATIENTS AND METHODS: Family-SCOUT represents a multicentric, prospective, interventional, and controlled study for families with parental cancer and their minor children. In the intervention group (IG), all family members were addressed using a care and case management approach for nine months. Families in the control group (CG) received standard of care. Participating parents were asked to complete the Hospital-Anxiety-Depression-Scale (HADS) questionnaire at enrolment (T0) and after 9 months (T2). The primary outcome was a clinically relevant reduction of distress in at least one parent per family, measured as minimal important difference (MID) of ≥1.6 in the HADS total score. The percentage of families achieving MID is compared between the IG and CG by exact Fisher's test, followed by multivariate confounder analyses. RESULTS: T0-questionnaire of at least one parent was available for 424 of 472 participating families, T2-questionnaire after 9 months was available for 331 families (IG n = 175, CG n = 156). At baseline, both parents showed high levels of distress (HADS total: sick parents IG: 18.7 ± 8.1; CG: 16.0 ± 7.2; healthy partners: IG: 19.1 ± 7.9; CG: 15.2 ± 7.7). The intervention was associated with a significant reduction in parental distress in the IG (MID 70.4% in at least one parent) compared with the CG (MID 55.8%; P = 0.008). Adjustment for group differences from specific confounders retained significance (P = 0.047). Bias from other confounders cannot be excluded. CONCLUSIONS: Parental cancer leads to a high psychosocial burden in affected families. Significant distress reduction can be achieved through an optimized and structured care approach directed at the family level such as family-SCOUT.

[HIS bundle pacing : Far more than an ECG cosmetic effect]. / HIS-Bundle-Pacing : Weit mehr als EKG-Kosmetik.

The therapy for heart failure in patients with uncompromised systolic ventricular function (HfpEF) is still challenging because there is an obvious lack of effective therapy options. Several of these particular patients are additionally presenting atrioventricular (AV) block. In these patients HIS bundle pacing could be a hopeful therapy strategy due to the option of an AV resynchronisation as illustrated in the following case.

Stillbirth: the impact of antiphospholipid syndrome?

Fetal death resulting in stillbirth is generally acknowledged as a feature of antiphospholipid syndrome. Recently published studies appear to confirm the association between antiphospholipid antibodies (aPL) and stillbirth, though additional studies of better design would be welcome. Emerging evidence suggests that treatment with heparin agents and low dose aspirin to prevent fetal death is imperfect. New therapeutic approaches for patients with lupus anticoagulant or triple aPL positivity are needed.

Leptin and leptin receptor polymorphisms and recurrent pregnancy loss.

OBJECTIVE: Leptin signaling is important in the establishment of pregnancy. We sought to determine if single nucleotide polymorphisms (SNPs) in the leptin and leptin receptor genes are associated with idiopathic recurrent pregnancy loss (RPL). STUDY DESIGN: We conducted a case-control study with cases defined as women with idiopathic RPL and controls as parous women without pregnancy losses. A total of 99 cases and 108 controls were genotyped for the leptin (-2548 G/A) SNP and the leptin receptor A223G SNP. Genotype and allele frequencies were compared between cases and controls using χ(2) test. RESULT: In this population, there was no significant difference in the genotype or allele frequencies for the leptin (-2548 G/A) or leptin receptor A223G SNPs between women with idiopathic RPL and controls. CONCLUSION: Although leptin signaling is critical to many aspects of reproduction, the maternal leptin and leptin receptor SNPs evaluated in this study are unlikely to have a clinically meaningful role in RPL.

Bait acceptability for delivery of oral rabies vaccine to free-ranging dogs on the Navajo and Hopi Nations.

In many areas of the world, only 30 to 50% of dogs are vaccinated against rabies. On some US Indian Reservations, vaccination rates may be as low as 5 to 20%. In 2003 and 2004, we evaluated the effectiveness of commercially available baits to deliver oral rabies vaccine to feral and free-ranging dogs on the Navajo and Hopi Nations. Dogs were offered one of the following baits containing a plastic packet filled with placebo vaccine: vegetable shortening-based Ontario slim baits (Artemis Technologies, Inc.), fish-meal-crumble coated sachets (Merial, Ltd.), dog food polymer baits (Bait-Tek, Inc.), or fish meal polymer baits (Bait-Tek, Inc.). One bait was offered to each animal and its behaviour toward the bait was recorded. Behaviours included: bait ignored, bait swallowed whole, bait chewed and discarded (sachet intact), bait chewed and discarded (sachet punctured), or bait chewed and consumed (sachet punctured). Bait acceptance ranged from 30.7% to 77.8% with the fish-meal-crumble coated sachets having the highest acceptance rate of the tested baits.

An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells.

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.

T-cell activation by recombinant immunoreceptors: impact of the intracellular signalling domain on the stability of receptor expression and antigen-specific activation of grafted T cells.

Recombinant immunoreceptors are modularily composed of extracellular antigen binding and intracellular signalling domains that are preferentially derived from CD3zeta or Fc epsilon RIgamma. The impact of the signalling domain on the stability of immunoreceptor expression and function is not completely understood. To address this issue, we generated and expressed a panel of recombinant zeta- and gamma-chain immunoreceptors, respectively, in human peripheral blood T cells. The expression level of zeta-chain immunoreceptors in human T cells is significantly lower than those of the homologous gamma-chain receptors. Low zeta-chain receptor expression in peripheral T cells is because of the intracellular signalling domain and independent of the Fc epsilon RIgamma or CD3zeta transmembrane region. Expression of both receptors decreases upon prolonged cultivation. Shortly after receptor engraftment, target cell lysis and induction of IFN-gamma secretion are mediated with similar efficiency by zeta- and gamma-chain immunoreceptors. Upon prolonged propagation, however, T-cell activation mediated by zeta-chain immunoreceptors is more efficient than by gamma-chain receptors, indicating that the initial high expression level of gamma-chain immunoreceptors compensates its lower activation capacity. Consequently, gamma-chain immunoreceptors exhibit a higher threshold value for specific activation and are more pronouncedly inhibited by soluble ligand antigen compared to the homologous zeta-chain receptor. These findings have substantial consequences for the design of recombinant immunoreceptors for use in adoptive immunotherapy.

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), CD3zeta (BW431/26-scFv-Fc-CD3zeta), or FcepsilonRIgamma (BW431/26-scFv-Fc-gamma). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcepsilonRIgamma or CD3zeta signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-gamma 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 gamma- or zeta-signaling receptors, but enhanced both IFN-gamma secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-gamma 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 CD3zeta signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3zeta) with dual signaling properties. T cells grafted with the combined CD28/CD3zeta 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.

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.

T cells engrafted with a recombinant anti-CD30 receptor target autologous CD30(+) cutaneous lymphoma cells.

T cells can be directed to antigen-specific, MHC-independent target cell 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(-) CD4(+) CD30(+) phenotype and clonal TCR-Vbeta7 rearrangement were isolated from a cutaneous lesion. The lymphoma lesion additionally harbored CD3(+) CD25(+) activated normal T cells despite ongoing tumor progression. Peripheral blood-derived T cells from the lymphoma patient were retrovirally engrafted with a recombinant anti-CD30-scFv-gamma receptor. Upon cocultivation with autologous CD30(+)lymphoma cells, grafted T cells increase IFN-gamma 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-gamma receptor overcomes T cell tolerance for tumor cells and directs T cells specifically against autologous lymphoma cells.

T-cell activation by recombinant receptors: CD28 costimulation is required for interleukin 2 secretion and receptor-mediated T-cell proliferation but does not affect receptor-mediated target cell lysis.

Recombinant T-cell receptors with antibody-like specificity are successfully used to direct CTLs toward a MHC-independent immune response against target cells. Here we monitored the specific activation of receptor grafted CTLs in the context of CD28 costimulation. Peripheral blood T cells were retrovirally engrafted with recombinant anti-CD30 and anti-carcinoembryonic antigen receptors, respectively, that harbor either the Fc epsilonRI-gamma or the CD3-zeta intracellular signaling domain. Cross-linking of recombinant receptors by solid-phase bound ligand, i.e., CD30 and a carcinoembryonic antigen receptor-specific anti-idiotypic antibody, respectively, induces IFN-gamma secretion that is further enhanced by CD28 costimulation of grafted T cells. Induction of interleukin (IL)-2 secretion, in contrast, requires CD28 costimulation in addition to receptor cross-linking, irrespective of T-cell preactivation by anti-CD3 monoclonal antibody plus IL-2 or by anti-CD3 monoclonal antibody plus anti-CD28 monoclonal antibody. Accordingly, induction of IL-2 secretion upon receptor cross-linking by membrane-bound antigen requires CD28/B7 costimulation whereas IFN-gamma secretion and cell proliferation does not. The efficiency of cytolysis by receptor-grafted CTLs does not depend on and is not affected by CD28 costimulation. The data demonstrate that CTL proliferation, cytokine secretion, and cytolysis upon receptor cross-linking are differentially modulated by CD28 costimulation and that cytolysis does not require B7 expression on target cells.

Dermal fibroblasts sustain proliferation of activated T cells via membrane-bound interleukin-15 upon long-term stimulation with tumor necrosis factor-alpha.

In chronic inflammatory conditions, mononuclear cells infiltrate the connective tissue attracted by fibroblast-secreted chemokines. The role of fibroblasts in sustaining the lymphocyte immune response upon cellular infiltration is so far unresolved. We here report that, upon prolonged stimulation with tumor necrosis factor-alpha, dermal fibroblasts enhance proliferation of activated T cells whereas unstimulated fibroblasts do not. T cell growth stimulation requires cell contact of tumor necrosis factor-alpha stimulated fibroblasts to T cells and is not due to soluble factors. Growth stimulation is substantially blocked by neutralizing antibodies to interleukin-15. Fluorescence-activated cell sorter analyses revealed that tumor necrosis factor alpha stimulated fibroblasts expose interleukin-15 in a membrane-bound form on the cell surface whereas nonstimulated fibroblasts and interferon-gamma treated fibroblasts do not. The amount of membrane interleukin-15 increases with the duration of tumor necrosis factor-alpha stimulation for at least 3 d. Unstimulated fibroblasts, however, accumulate interleukin-15 in the cytoplasm. No interleukin-15 could be detected in the culture supernatant. Immunohistochemical analyses confirmed membrane interleukin-15 on dermal fibroblasts in discoid lupus erythematosus skin lesions whereas no membrane interleukin-15 was found on the surface of fibroblasts in healthy skin. We conclude that dermal fibroblasts upon long-term tumor necrosis factor-alpha stimulation during chronic inflammation are involved via membrane-bound interleukin-15 in stimulating proliferation of accumulated, activated T cells.

A novel strategy in the elimination of disseminated melanoma cells: chimeric receptors endow T cells with tumor specificity.

The application of immunotherapy to the treatment of micrometastases of melanoma has attracted growing interest in recent years. This trend reflects, at least in part, the disappointing results of conventional chemotherapy, the identification of melanoma-associated antigens suitable to be used as targets for immunotherapy, and the significant progress in our understanding of molecular processes involved in the development of an immune response. Because of the general belief that T cell immunity plays a major part in the control of tumor growth, we have recently applied a novel strategy to target cytolytic T cells to melanoma cells. The strategy bypasses the requirement of presentation of melanoma-associated-antigen-derived peptides by the major histocompatibility complex to the T cell receptor complex by permanent grafting of T cells with a recombinant, chimeric T cell receptor. The extracellular moiety of the grafted receptor contains the antigen-binding domain, consisting of a single-chain antibody fragment (scFv) derived from a monoclonal antibody specific for the high-molecular-weight melanoma-associated antigen (HMW-MAA). The intracellular receptor moiety contains the cellular activation domain, consisting of the gamma-signaling chain derived from the Fc epsilon RI receptor. Cytotoxic T cells grafted with the chimeric anti-HMW-MAA scFv-gamma signaling receptor specifically lyse HMW-MAA-positive melanoma cells in a human leukocyte antigen class I-independent fashion. The chimeric T cell receptor strategy is designed to eliminate disseminated tumor cells by the power of cytolytic T cells that physiologically penetrate tissues and that are specifically activated by the grafted receptor after binding to antigen-positive melanoma cells.

Enhanced green fluorescent protein fusion proteins of herpes simplex virus type 1 thymidine kinase and cytochrome P450 4B1: applications for prodrug-activating gene therapy.

To monitor therapeutic transgene expression, we developed fusion genes of enhanced green fluorescent protein (EGFP) with two different prodrug-activating enzyme genes: herpes simplex virus type 1 thymidine kinase (HSV-tk) and rabbit cytochrome P450 4B1 (cyp4b1). Expression of the resulting fusion proteins, TK-EGFP and 4B1-EGFP, rendered transduced human and rodent glioma cells sensitive to cytotoxic treatment with the corresponding prodrugs ganciclovir and 4-ipomeanol. Ganciclovir and 4-ipomeanol sensitivity was comparable with that achieved with the native HSV-TK and CYP4B1 proteins. As shown by fluorescence microscopy, TK-EGFP was expressed predominantly intranuclearly, whereas 4B1-EGFP was detectable in the cytoplasm, thereby displaying the orthotopic subcellular distribution of the corresponding native enzymes. The fluorescence intensity correlated well with the corresponding prodrug sensitivity, as shown by fluorescence-activated cell sorter analysis. EGFP expression was also used for the selection of stably HSV-tk-transduced cells by flow cytometric cell sorting. Resulting cell populations showed a homogeneity of fluorescence intensity similar to single-cell clones after antibiotic selection. In conclusion, tk-egfp and 4b1-egfp fusion genes are valuable tools for monitoring prodrug-activating gene therapy in living cells. EGFP fusion genes/proteins provide a simple and reproducible means for the detection, selection, and characterization of cells expressing enzyme genes for prodrug activation.

T cell activation by recombinant FcepsilonRI gamma-chain immune receptors: an extracellular spacer domain impairs antigen-dependent T cell activation but not antigen recognition.

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 (anti-CD30 HRS3-scFv) and signaling domain (FcepsilonRI gamma-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 spacer-less 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.

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 Hodgkin's 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 Hodgkin's disease.

A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA.

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 gamma-signaling chain of the human Fc epsilon RI receptor constituting a completely humanized chimeric receptor. After transfection, the humBW431/26 scFv-CH2CH3-gamma 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 micrograms/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.

Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor.

Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor.

Chimeric anti-TAG72 receptors with immunoglobulin constant Fc domains and gamma or zeta signalling chains.

We recently described the generation and expression of a chimeric T cell receptor with specificity for the tumor antigen TAG72 consisting of the single chain antibody (scFv) B72.3-scFv and the gamma chain of the FcepsilonRI receptor. The corresponding chimeric receptor containing the zeta chain of the TCR as signalling unit is not functionally expressed reflecting that the requirements for functional expression of chimeric receptors containing the gamma signalling chain are apparently different compared to those containing the CD3zeta signalling chain of the TCR. We describe a novel set of chimeric anti-TAG72 receptors including in their extracellular moiety the constant immunoglobulin CH2/3 domains that allow stable expression of chimeric gamma as well as zeta receptors. We designed anti-TAG72 receptors that consist of a scFv fragment derived from an anti-TAG72 second generation antibody (CC49) and of the CH2/3 domains of the human IgG and intracellularily either of the zeta or gamma signalling chain. The recombinant CC49-CH2/3-zeta and CC49-CH2/3-gamma DNA, respectively, was transfected into MD45 T cells and expressed under control of the RSV LTR. Both receptors were found on the cell membrane of transfected cells as demonstrated by flow cytometry analysis using an anti-human IgG Fc antibody directed to the CH2/3 immunoglobulin domains of the chimeric receptor. Specific cross-linking of the chimeric zeta as well as the gamma receptor by antigen or anti-human Ig antibodies resulted in specific activation of transfected cells. Our results demonstrate that both the gamma chain and the zeta chain++ containing receptor are stably expressed and convert T cells to specificity for the TAG72 antigen. This receptor design will facilitate efficient generation of genetically modified peripheral T cells and may provide valuable tools for the cellular immunotherapy of TAG72+ tumors.