A dual chain chimeric antigen receptor (CAR) in the native antibody format for targeting immune cells towards cancer cells without the need of an scFv.

Adoptive cell therapy with chimeric antigen receptor (CAR)-modified T cells showed remarkable therapeutic efficacy in the treatment of leukaemia/lymphoma. However, the application to a variety of cancer entities is often constricted by the non-availability of a single chain antibody (scFv), which is usually the targeting domain in a CAR, while antibodies in the natural format are often available. To overcome the limitation, we designed a CAR that uses an antibody in its natural configuration for binding. Such CAR consists of two chains, the immunoglobulin light and heavy chain with their constant regions, whereby the heavy chain is anchored to the membrane and linked to an intracellular signalling domain for T-cell activation. The two chains form a stable heterodimer, a so-called dual chain CAR (dcCAR), and bind with high affinity and in a specific manner to their cognate antigen. By specific binding, the dcCAR activates engineered T cells for the release of pro-inflammatory cytokines and for target cell lysis. We provide evidence by three examples that the dcCAR format is universally applicable and thereby broadens the CAR cell therapy towards a larger variety of targets for which an scFv antibody is not available.

No stress--Hsp90 and signal transduction in Leishmania.

SUMMARY Hsp90 (a.k.a. Hsp83) plays a significant role in the life cycle control of the protozoan parasite Leishmania donovani. Rather than protecting Leishmania spp. against adverse and stressful environs, Hsp90 is required for the maintenance of the motile, highly proliferative insect stage, the promastigote. However, Hsp90 is also essential for survival and proliferation of the intracellular mammalian stage, the amastigote. Moreover, recent evidence shows Hsp90 and other components of large multi-chaperone complexes as substrates of stage-specific protein phosphorylation pathways, and thus as likely effectors of the signal transduction pathways in Leishmania spp. Future efforts should be directed towards the identification of the protein kinases and the critical phosphorylation sites as targets for novel therapeutic approaches.

The weal and woe of costimulation in the adoptive therapy of cancer with chimeric antigen receptor (CAR)-redirected T cells.

Adoptive cell therapy has shown impressive efficacy to combat cancer in early phase clinical trials, in particular when T cells engineered to specifically target tumor cells were applied. The patient's T cells are genetically equipped with a chimeric antigen receptor (CAR) which allows them to be redirected in a predefined manner towards virtually any target; by using an antibody-derived domain for binding, CAR T cells can be redirected in a major histocompatibility complex (MHC) dependent and independent fashion. The CAR also provides the stimuli required to induce and maintain T cell activation. Recent clinical data sustain the notion that strong costimulation in conjunction with the primary activation signal is crucial for lasting therapeutic efficacy of CAR T cells. However, costimulation is a double-edged sword and the impact of the individual costimuli to optimize T cell activation is still under debate; some general rules are emerging. The review summarizes how costimulation modulates, improves and prolongs the redirected anti-tumor T cell response and how the same costimulatory signals may contribute to unintended side effects including "cytokine storm" and T cell repression. Upcoming strategies to break the activation/repression circle by using CAR's with modified costimulatory signals are also discussed.

T cells redirected by a CD3ζ chimeric antigen receptor can establish self-antigen-specific tumour protection in the long term.

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.

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack.

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.

[Adoptive T-cell therapy of rhabdomyosarcoma]. / Adoptive T-Zell-Therapie des Rhabdomyosarkoms.

AIMS: To improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen. METHODS: A "second generation" chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis. RESULTS: Co-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the "second generation" AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR. CONCLUSIONS: Data suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.

Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc 'spacer' domain in the extracellular moiety of chimeric antigen receptors avoids 'off-target' activation and unintended initiation of an innate immune response.

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.

Redirecting human CD4+CD25+ regulatory T cells from the peripheral blood with pre-defined target specificity.

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.

Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer.

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-gamma (IFNgamma), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNFalpha). 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.

A recombinant anti-carcinoembryonic antigen immunoreceptor with combined CD3zeta-CD28 signalling targets T cells from colorectal cancer patients against their tumour cells.

BACKGROUND AND AIMS: The prognosis of metastatic colorectal cancer is still poor, raising the need for alternative therapeutic approaches, particularly by manipulating the antitumour immune response. Advanced tumour stages, however, are frequently accompanied by functional T cell defects which may be critical for a T cell based anticancer immunotherapy. The aim of this study was to address whether T cells from colorectal cancer patients with advanced tumour stages can be specifically antigen activated against their autologous tumour cells. METHODS: T cells were isolated from colorectal cancer patients and retrovirally transduced to express a recombinant immunoreceptor that has an extracellular binding domain for carcinoembryonic antigen (CEA) and an intracellular CD3zeta signalling domain with and without CD28 costimulation for T cell activation. RESULTS: Peripheral blood T cells from colorectal cancer patients were successfully engineered to express the anti-CEA immunoreceptor on the cell surface. On coincubation with autologous CEA(+) tumour cells, T cells with anti-CEA immunoreceptor are specifically activated to secrete interferon gamma (IFN-gamma) and to lyse autologous tumour cells whereas T cells without immunoreceptor are not. T cells equipped with combined CD3zeta-CD28 signalling receptor are more efficiently activated to secrete IFN-gamma compared with T cells with CD3zeta signalling receptor. Induction of interleukin 2 secretion on targeting towards autologous tumour cells requires triggering of T cells by the CD3zeta-CD28 costimulatory receptor. CONCLUSIONS: T cells from advanced colorectal cancer patients can be tumour specifically activated with high efficiency by engraftment with a combined CD3zeta-CD28 immunoreceptor to break tolerance against autologous tumour cells.

An interleukin 12 p40-IgG2b fusion protein abrogates T cell mediated inflammation: anti-inflammatory activity in Crohn's disease and experimental colitis in vivo.

BACKGROUND AND AIMS: Interleukin-12 (IL-12), a p35/p40 heterodimer, plays a pivotal role in the immune response in Crohn's disease (CD). Since IL-12 p40 dimers act as IL-12 antagonists, we assayed p40 dimer proteins to modulate chronic intestinal inflammation. METHODS: We generated a fusion protein consisting of the IL-12(p40) subunit fused to the constant region of IgG2b. IL-12(p40)-IgG2b was tested in a murine 2,4,6,-trinitrobenzene sulphonic acid (TNBS) colitis model and in lamina propria mononuclear cells (LPMNC) from patients with CD in vitro. RESULTS: Dimeric IL-12(p40)-IgG2b fusion protein bound specifically to the IL-12 receptor. In concentrations <10(-7) M, it acted as an IL-12 antagonist as it inhibited interferon gamma (IFN-gamma) secretion, suppressed proliferation, and increased apoptosis of LPMNC from patients with CD. However, in concentrations >10(-6) M, IL-12(p40)-IgG2b increased IFN-gamma secretion and lymphocyte proliferation thereby acting as an IL-12 agonist. In TNBS colitic mice, IL-12(p40)-IgG2b decreased mortality (10% v 68%), prevented body weight loss, reduced tumour necrosis factor alpha, and increased IL-10 secretion. CONCLUSIONS: The IL-12(p40)-IgG2b fusion protein has dichotomic properties as a specific IL-12 antagonist and selective repressor of mucosal inflammation at low concentration and as an IL-12 agonist at high concentration.

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.

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.

An entirely humanized CD3 zeta-chain signaling receptor that directs peripheral blood t cells to specific lysis of carcinoembryonic antigen-positive tumor cells.

Recombinant T-cell receptors with antibody-like specificity for tumor-associated antigens are successfully used to direct the cytolytic activity of T cells toward tumor cells. Clinical application, however, needs to comply with the low immunogenicity of the recombinant receptor, efficient gene transfer into peripheral blood T cells, and enrichment of receptor-grafted cells. Here, we address these issues and describe an entirely humanized immune receptor for use in adoptive immunotherapy of colorectal carcinoma. The receptor consists of a single-chain antibody (scFv) binding domain specific for carcinoembryonic antigen (CEA), the IgG hinge and CH2/CH3 (Fc) joining region, and the transmembrane and intracellular CD3 zeta signaling chain. To express the receptor in peripheral blood T cells, both GALV envelope and MuLV 4070A pseudotyped retrovirus turned out to be equally efficient, with transduction efficiencies of about 5% to 40%, depending on the lymphocyte donor. Furthermore, receptor-grafted T cells could be 2- to 6-fold enriched by magnetic activated cell sorting, utilizing an antibody directed to the extracellular IgG domain of the receptor. Upon co-culture with CEA(+) tumor cells, receptor-grafted T cells are specifically and efficiently activated to cytolysis and IFN-gamma secretion, demonstrating their feasibility for the adoptive immunotherapy of CEA(+) carcinomas.