A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

To harness the potent tumor-killing capacity of T cells for the treatment of CD19⁺ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19⁺ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19⁺ malignancies with an advantageous safety risk profile and anticipated dosing regimen.     

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

To harness the potent tumor-killing capacity of T cells for the treatment of CD19⁺ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19⁺ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19⁺ malignancies with an advantageous safety risk profile and anticipated dosing regimen.     

CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms

The glucocorticoid-induced TNF-related gene receptor (GITR) is the newest member of the costimulatory molecule family and is expressed on both resting CD4+CD25+ regulatory T (T(R)) cells and activated CD4+ T cells. We investigated the endogenous mechanisms that regulate GITR expression on both T(R) and CD4+ T cells, as well as the functional interaction between GITR and other costimulatory molecules. CD28 stimulation increased GITR expression on both T(R) and CD4+ T cells via IL-2-dependent mechanisms. In addition, ligation of GITR and/or CD28 increased the level of CD4+ T cell proliferation and effector function under both APC-dependent and -independent conditions, suggesting that these costimulatory molecules cooperate to regulate CD4+ T cell activation and function by directly signaling to the CD4+ T cell. Thus, GITR may serve opposing functional roles on CD4+ T(R) and effector cells and alterations in GITR expression and/or function may tip the balance between immune tolerance and effector function.     

CD103 is a marker for alloantigen-induced regulatory CD8+ T cells

The alphaEbeta7 integrin CD103 may direct lymphocytes to its ligand E-cadherin. CD103 is expressed on T cells in lung and gut and on allograft-infiltrating T cells. Moreover, recent studies have documented expression of CD103 on CD4+ regulatory T cells. Approximately 4% of circulating CD8+ T cells bear the CD103 molecule. In this study, we show that the absence or presence of CD103 was a stable trait when purified CD103- and CD103+ CD8+ T cell subsets were stimulated with a combination of CD3 and CD28 mAbs. In contrast, allostimulation induced CD103 expression on approximately 25% of purified CD103- CD8+ T cells. Expression of CD103 on alloreactive cells was found to be augmented by IL-4, IL-10, or TGF-beta and decreased by addition of IL-12 to MLCs. The alloantigen-induced CD103+ CD8+ T cell population appeared to be polyclonal and retained CD103 expression after restimulation. Markedly, in vitro-expanded CD103+ CD8+ T cells had low proliferative and cytotoxic capacity, yet produced considerable amounts of IL-10. Strikingly, they potently suppressed T cell proliferation in MLC via a cell-cell contact-dependent mechanism. Thus, human alloantigen-induced CD103+ CD8+ T cells possess functional features of regulatory T cells.     

Potent inhibition of local and disseminated tumor growth in immunocompetent mouse models by a bispecific antibody construct specific for Murine CD3

Bispecific single-chain antibody constructs specific for human CD3 have been extensively studied for antitumor activity in human xenograft models using severe combined immunodeficient mice supplemented with human T cells. High efficacy at low effector-to-target ratios, independence of T cell costimuli and a potent activation of previously unstimulated polyclonal T cells were identified as hallmarks of this class of bispecific antibodies. Here we studied a bispecific single-chain antibody construct (referred to as ‘bispecific T cell engager’, BiTE) in an immunocompetent mouse model. This was possible by the use of a murine CD3-specific BiTE, and a syngeneic melanoma cell line (B16F10) expressing the human Ep-CAM target. The murine CD3-specific BiTE, called 2C11x4-7 prevented in a dose-dependent fashion the outgrowth of subcutaneously growing B16/Ep-CAM tumors with daily i.v. injections of 5 or 50 μg BiTE which was most effective. Treatment with 2C11x4-7 was effective even when it was started 10 days after tumor cell inoculation but delayed treatments showed a reduction in the number of cured animals. 2C11x4-7 was also highly active in a lung tumor colony model. When treatment was started on the day of intravenous tumor cell injection, seven out of eight animals stayed free of lung tumors, and three out of eight animals when treatment was started on day 5. Our study shows that BiTEs also have a high antitumor activity in immunocompetent mice and that there is no obvious need for costimulation of T cells by secondary agents. Bernd Schlereth and Petra Kleindienst contributed equally to this work.     

Expression and function of the B and T lymphocyte attenuator (BTLA/CD272) on human T cells

Co-signal receptors provide crucial activating or attenuating signals for T cells. The B and T lymphocyte attenuator (BTLA/CD272) is a third member of co-inhibitory receptors, which belongs to the CD28 immunoglobulin-superfamily. Using monoclonal antibodies (mAbs) against human BTLA, we show that BTLA is constitutively expressed on most CD4+ and CD8+ T cells and its expression progressively decreases upon T cell activation. Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations, but the extended culture diminished BTLA expression. Cross-linking BTLA with an agonistic mAb inhibited T cell proliferation and the production of the cytokines IFN-gamma and IL-10 in response to anti-CD3 stimulation. BTLA-mediated inhibition of T cell activation occurred during both primary CD4+ T cell responses and secondary CD4+ and CD8+ T cell responses, suggesting that BTLA ligation sends a constitutive "off" signal to T cells and thus might play an important role in the maintenance of T cell tolerance.     

Early kinetic window of target T cell susceptibility to CD25+ regulatory T cell activity

Peripheral tolerance is maintained in part by thymically derived CD25+CD4+ T cells (regulatory T cells (Tregs)). Their mechanism of action has not been well characterized. Therefore, to get a better understanding of Treg action, we investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6-10 h of culture. Visualization of this time frame, using a sensitive single-cell assay for IL-2, revealed the early elaboration of target cell IL-2 producers in the first 6 h despite the presence of CD25+CD4+ Tregs. However, after 6 h, a rapid rise in the number of IL-2 producers in the absence of Tregs was dramatically abrogated by the presence of Tregs. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. Modulating target T cell activation signals with provision of CD28, IL-2, or high Ag dose all abrogated suppression of proliferation late in culture. However, only CD28 signals enabled target T cells to resist the early Treg-induced down-regulation of IL-2. Therefore the quality of early target T cell activation signals, in particular engagement of CD28, represents an important control point in the balance between vulnerability and resistance to Treg suppression.     

HIV-1 Nef induces dendritic cell differentiation: a possible mechanism of uninfected CD4(+) T cell activation

Human immunodeficiency virus (HIV)-1 Nef protein is an essential modulator of AIDS pathogenesis and we have previously demonstrated that rNef enters uninfected human monocytes and induces T cells bystander activation, up-regulating IL-15 production. Since dendritic cells (DCs) play a central role in HIV-1 primary infection we investigated whether rNef affects DCs phenotypic and functional maturation in order to define its role in the immunopathogenesis of AIDS. We found that rNef up-regulates the expression on immature DCs of surface molecules known to be critical for their APC function. These molecules include CD1a, HLA-DR, CD40, CD83, CXCR4, and to a lower extent CD80 and CD86. On the other hand, rNef down-regulates surface expression of HLA-ABC and mannose receptor. The functional consequence of rNef treatment of immature DCs is a decrease in their endocytic and phagocytic activities and an increase in cytokine (IL-1beta, IL-12, IL-15, TNF-alpha) and chemokine (MIP-1alpha, MIP-1beta, IL-8) production as well as in their stimulatory capacity. These results indicate that rNef induces a coordinate series of phenotypic and functional changes promoting DC differentiation and making them more competent APCs. Indeed, Nef induces CD4(+) T cell bystander activation by a novel mechanism involving DCs, thus promoting virus dissemination.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

A regulatory role for the CD4 and CD8 molecules in T cell activation

The role of the CD4 and CD8 molecules in T cell activation is presently a matter of controversy. Although their role as associative binding elements to MHC class II or class I is well documented, their influence on the triggering process in unclear. Because antibodies to CD4 or CD8 block T cell activation in the absence of their respective ligands, a negative signaling by these molecules has been suggested. However, recent experimental evidence argues against a negative regulatory effect of these molecules, since, e.g., simultaneous cross-linking of TCR and CD4 leads to enhanced T cell activation. Therefore, a current model suggests that the association of TCR and CD4 in the membrane gives a positive signal essential for triggering. In this report we present evidence that this model is likely to be too simple. Anti-CD4 and CD8 antibodies inhibit alternative, nonreceptor pathways of T cell triggering via Tp103 and Tp44 in the absence of class II positive accessory or target cells. These antibodies also inhibit bypass activation of T cells by phorbol ester and calcium ionophore in an accessory cell-free system. Furthermore, if the CD4 or CD8 molecules are removed from the cell surface by antibody-induced modulation, the proliferative and cytotoxic response of T cell clones is enhanced. This enhancement is also observed if resting peripheral blood T cells are used as responder cells. These data show that the CD4 or CD8 molecules have a complex regulatory function in T cell activation beyond the requirement for co-cross-linking with the TCR.     

Triggering of cytotoxic T lymphocytes and NK cells via the Tp103 pathway is dependent on the expression of the T cell receptor/CD3 complex

The expression and function of the T cell activation molecule Tp103 on human cloned cytotoxic CD3+ and CD3- cells were studied. All in vitro growing CD3+ and CD3- clones expressed Tp103 regardless of their phenotype and the expression of a CD3-associated TCR complex. Whereas the CD2 pathway was functional in all these clones, only CD3-expressing clones could be triggered via Tp103 to kill target cells. In contrast, both CD2 and Tp103 pathways were suppressed after modulation of the TCR complex with anti-CD3 mAb. This indicates that the function of Tp103 but not of CD2 is dependent on the expression of a functional Ag receptor on cytotoxic T cells. Furthermore, modulation of the Ag receptor induces a state of unresponsiveness in cytotoxic T cells that cannot be attributed to just the removal of the CD3/TCR complex from the cell membrane.     

Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.     

CD4 T cell dependent tumor immunity stimulated by dendritic cell based vaccine

CD8 CTLs have been accountable for the major effector cells responsible for the rejection of tumor cells. And CD40 signaling and IL-12 have been shown to be the essential pathways involved in the activation process. Immunizing mice with dendritic cells transduced with an adenovirus expressing the human melanoma antigen gp100, an immunization strategy of xenoimmunization, stimulated potent tumor protection dependent on effective CD4 T cells in the absence of CD8 T cells. Further studies revealed that neither CD40 signaling nor IL-12 was indispensable for the activation of dendritic and CD4 T cells in this model. Stimulation of effective antitumor immunity targeting the self-antigen did not elicit autoimmunity. The implications of this study were discussed.     

口服卡介菌诱导免疫耐受的CD4+CD25+调节性T 细胞机制研究

目的:研究口服卡介菌诱导免疫耐受对CD4+CD25+调节性T细胞的影响。方法:采用口服MPB制备EAE大鼠模型,随机分为BCG组(0.5mg/kg)和EAE模型组(PBS),每组各15只,连续经口灌服给药14d,同时选取15只健康大鼠作为对照组。分别于免疫后15d、27d流式细胞术检测外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率,ELISA检测血清IL-6、TGF-β、IgE、IgG含量。结果:与EAE模型组相比,免疫后BCG组大鼠外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率增加,血清IL-6、TGF-β含量上升,血清IgE、IgG抗体水平下降。结论:口服BCG通过上调淋巴器官中CD4+CD25+T淋巴细胞比例,抑制效应性T细胞活性,发挥免疫耐受作用。     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.