The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3x10(-7) M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.     

Cytokine-induced killer cells targeted by the novel bispecific antibody CD19xCD5 (HD37xT5.16) efficiently lyse B-lymphoma cells

Due to their dual binding capacity, bispecific antibodies (bsAb) can be used to cross-link cytotoxic effector cells with malignant targets and may thereby improve adoptive immunotherapy. In this study, the development and preclinical testing of the quadroma-derived bsAb HD37xT5.16 of the specificity CD19xCD5 is reported. Effector cells used were a population of ex vivo expanded and activated T cells called cytokine-induced killer (CIK) cells expressing CD5. When combined with CIK cells, the cytolytic potency of HD37xT5.16 against CD19 positive B cell lymphoma lines was comparable to that observed with a previously described CD19xCD3 bsAb. Further on, we could demonstrate that bsAb CD19xCD5, in contrast to its CD3-binding counterpart, does not induce proliferation of resting T cells and causes only little activation-induced cell death. Therefore, this novel bsAb binding effector T cells via CD5 may be particularly useful in combination with adoptive transfer of ex vivo activated T cells, e.g., in the setting of adoptive immunotherapy after allogeneic stem cell transplantation. The in vitro studies outlined here support the experimental use of bsAb HD37xT5.16 in preclinical in vivo models for evaluation of its safety and efficacy profile. Freddy Tita-Nwa and Gerhard Moldenhauer contributed equally to the study and share first authorship.     

A novel bispecific single-chain antibody for ADAM17 and CD3 induces T-cell-mediated lysis of prostate cancer cells

ADAM17 (A disintegrin and metalloproteinase 17) is a membrane-bound protease that cleaves various cell surface proteins, including cytokines and cytokine receptors. Recently it was shown that ADAM17 is highly expressed on the surface of many cancer cells, whereas normal cells express low levels of ADAM17, implying that ADAM17 is a potential immunotherapeutic target. We have generated a monoclonal antibody against human ADAM17, which recognized the membrane proximal cysteine-rich extension of the ADAM17 protein. Unlike normal cells, tumour cell lines, such as a prostate cancer cell line, pancreatic cancer cell lines, a breast cancer cell line and a non-small lung cancer cell line, expressed ADAM17 on the cell surface. Using the sequence of the antibody we generated an ADAM17-specific scFv (single-chain variable fragment) and fused this to a CD3-specific scFv to generate a bispecific T-cell engager antibody [A300E-BiTE (bispecific T-cell engager antibody)]. Specificity was demonstrated on cells in which ADAM17 was knocked down with a specific shRNA (short hairpin RNA). A300E-BiTE recognized ADAM17 and CD3 on the cell surface of tumour cells and T-cells respectively. In the presence of primary human peripheral blood mononuclear cells or human T-cells the addition of A300E-BiTE led to ADAM17-specific killing of prostate tumour cells indicating a novel strategy for the treatment of cancer.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997     

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.     

Induction of antitumor immunity after cure of pulmonary metastases, using staphylococcal enterotoxin B and bispecific antibody

The combination of staphylococcal enterotoxin B (SEB) and anti-p97 x anti-CD3 bispecific antibody (bsAb) cures 60%-80% of mice with established pulmonary metastases of the syngeneic p97+ murine melanoma, CL62. We investigated the ability of cured mice to generate protective antitumor immunity. In tumor rechallenge experiments, CL62-cured mice developed protective immunity against rechallenge with CL62. The majority of mice also rejected the p97-negative parental cell line, K1735, indicating an immune response to tumor antigens common to both cell lines that were not bsAb-targeted. A significant humoral response developed against p97 antigen, but not against other antigens common to both CL62 and K1735. That the majority of cured mice nevertheless rejected K1735 suggests that tumor immunity is not antibody-dependent. Evidence of cellular immunity was obtained from the results of delayed-type hypersensitivity, proliferation and cytotoxicity assays, which revealed the presence of tumor-specific memory in bsAb-treated, CL62-cured mice. CD8+ T cells from cured, but not control mice were able to lyse tumor; however, memory CD4 cells had no cytolytic function. In vivo, however, both CD4 and CD8 T cells were required for effective protective immunity. These studies demonstrate that treatment with SEB and bsAb not only confers passive immune effects of tumor eradication, but also actively promotes the generation of a host antitumor immune response.     

Lysis of murine B lymphoma cells by transgenic phagocytes via a human FcγRI×murine MHC class II bispecific antibody

 The class I IgG receptor (FcγRI) on cytotoxic effector cells has been reported to initiate destruction of tumour cells by effector cells in vitro. We are aiming at developing an immunocompetent model to evaluate the cytotoxic capacity of human FcγRI for the rejection of tumour cells in vivo. Therefore, we recently generated a transgenic mouse strain expressing human FcγRI on monocytes, macrophages, and neutrophils. In these mice, the human receptor is up-regulated by granulocyte-colony-stimulating factor (G-CSF) and is able to trigger cellular responses. Subsequently, in the present study the B cell lymphoma IIA1.6 cell line is selected as a tumour target, and a human FcγRI-directed antitumour bispecific antibody (bsAb) is constructed and characterized. Fab′ fragments of mAb 22, which bind hFcγRI at an epitope that is distinct from the ligand binding site, were chemically linked to Fab′ fragments of rat anti-(mMHC class II antigens) mAb M5/114, yielding bsAb 22×M5/114. This bsAb was able to bind simultaneously to hFcγRI and mMHC class II antigens in a dose-dependent fashion. Binding of 22×M5/114 to FcγRI was not inhibited in the presence of human IgG. It is important to note that, MHC-class-II-expressing IIA1.6 lymphoma cells were lysed by whole blood from G-CSF-treated transgenic mice in the presence of bsAb 22×M5/114. No lysis by whole blood from non-transgenic mice or from transgenic animals that had not received G-CSF was observed. These results indicate that human FcγRI is able to mediate lysis of murine IIA1.6 lymphoma cells by transgenic effector cells via bsAb 22×M5/114. A trial with transgenic mice, evaluating the efficacy of these hFcγRI-directed bsAb in combination with G-CSF for treatment of IIA1.6 B cell lymphoma, is currently in progress. Accepted: 14 October 1997     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

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.     

The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531

In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 × CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of⁵¹Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 × CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.     

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.     

Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells

 Bispecific monoclonal antibodies (bi-mAb), directed against a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes, induce activation of resting T lymphocytes and target-specific tumor cell lysis. We now show that both necrosis and apoptosis contribute to T-cell-mediated tumor cell destruction. Even though T cells up-regulate FAS/APO-1 expression upon bi-mAb stimulation, FAS/APO-1-mediated apoptosis does not contribute to bi-mAb-mediated destruction of Hodgkin’s cells. CD8⁺ lymphocytes were the most potent effectors of bi-mAb-mediated cytotoxicity and had the highest levels of mRNA coding for perforin and granzyme A and B. Ca²⁺-complexing agents, which abrogate perforin activity, led to decreased levels of necrosis, while inhibition of granzyme activity in effector or target cells had a similar effect on apoptosis. Granzyme-mediated apoptosis critically dependent on the proliferative state of the target cells, while perforin-induced necrosis was not cell-cycle-dependent. Our results underline the importance of the expression levels of perforin and granzymes in the effector T cells and of the proliferative state of the target cells in bi-mAb-mediated apoptosis and necrosis of tumor cells. Received: 5 December 1996 / Accepted: 16 January 1997     

Co-engaging CD47 and CD19 with a bispecific antibody abrogates B-cell receptor/CD19 association leading to impaired B-cell proliferation

CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.     

A comparative study of the bispecific monoclonal antibody,blinatumomab expression in CHO cells and E. coli

Abstract

The “bispecifics” market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6?×?His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS–PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55?kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.     

CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas

Individual or combinations of somatic mutations found in genes from colorectal cancers can redirect the effects of chemotherapy and targeted agents on cancer cell survival and, consequently, on clinical outcome. Novel therapeutics with mechanisms of action that are independent of mutational status would therefore fulfill a current unmet clinical need. Here the CEA and CD3 bispecific single-chain antibody MEDI-565 (also known as MT111 and AMG 211) was evaluated for its ability to activate T cells both in vitro and in vivo and to kill human tumor cell lines harboring various somatic mutations commonly found in colorectal cancers. MEDI-565 specifically bound to normal and malignant tissues in a CEA-specific manner, and only killed CEA positive cells. The BiTE® antibody construct mediated T cell-directed killing of CEA positive tumor cells within 6 hours, at low effector-to-target ratios which were independent of high concentrations of soluble CEA. The potency of in vitro lysis was dependent on CEA antigen density but independent of the mutational status in cancer cell lines. Importantly, individual or combinations of mutated KRAS and BRAF oncogenes, activating PI3KCA mutations, loss of PTEN expression, and loss-of-function mutations in TP53 did not reduce the activity in vitro. MEDI-565 also prevented growth of human xenograft tumors which harbored various mutations. These findings suggest that MEDI-565 represents a potential treatment option for patients with CEA positive tumors of diverse origin, including those with individual or combinations of somatic mutations that may be less responsive to chemotherapy and other targeted agents.     

Tumor-specific targeting of T helper type 1 (Th1) cells by anti-CD3 × anti-c-ErbB-2 bispecific antibody

T helper type1 (Th1) or type2 (Th2) cells were induced from naive Th cells obtained from ovalbumin-specific T cell receptor (TCR) transgenic mice. Th1 cells producing interferon γ (IFNγ) exhibited stronger antigen-specific cytotoxicity against ovalbumin-(323–339)-peptide-pulsed A20 tumor cells than did Th2 cells. To develop a general method for applying antigen-nonspecific Th1 cells to tumor immunotherapy, we examined the targeting of Th1 cells to tumor cells using a bispecific antibody (bsAb) consisting of anti-(mouse CD3) mAb and anti-(human c-ErbB-2) mAb. When ovalbumin-specific Th1 or Th2 cells were cocultured with c-erbB-2-positive transfectants (CMS7HE), neither type of cell showed significant cytotoxicity or cytokine production in response to tumor cells. However, addition of bsAb resulted in the triggering of both Th1 and Th2 cells. Th1 cells showed higher levels of bsAb-dependent cytotoxicity against CMS7HE tumor cells than did Th2 cells. The targeting of Th1 cells to CMS7HE tumor cells by bsAb also triggered the production of cytokines such as IFNγ, interleukin-2 and tumor necrosis factor α (TNFα). The released TNFα was demonstrated to be a critical cytolytic factor in bsAb-mediated cytotoxicity by Th1 cells. Finally, Th1 cells were demonstrated to show antitumor activity in vivo against human c-erbB-2-positive tumor cells implanted in nude mice. These results suggest that Th1 cells are useful effector cells for the application to adoptive tumor immunotherapy in conjunction with bsAb. Received: 22 April 1999 / Accepted: 2 July 1999     

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.     

CD4 co-receptor dependent signaling promotes competency for re-stimulation induced cell death of effector T cells

The elimination of activated T cells by FAS-mediated signaling is an important immunoregulatory mechanism used to maintain homeostasis and prevent tissue damage. T cell receptor-dependent signals are required to confer sensitivity to FAS-mediated re-stimulation-induced cell death (RICD), however, the nature of these signals is not well understood. In this report, we show, using T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene, that CD4-dependent signaling events are a critical part of the competency signal required for RICD. This is in part due to defects in FAS receptor signaling complex formation as shown by decreased recruitment of FAS and caspase 8 into lipid rafts following antigen re-stimulation in the absence of CD4-dependent signals. In addition, in the absence of CD4-dependent signals, effector T cells have a selective defect in IL-2 secretion following peptide re-stimulation, while provision of exogenous IL-2 during re-stimulation partially restores susceptibility to RICD. Importantly, IL-2 production and proliferation after primary peptide stimulation is comparable between wild type and CD4-deficient T cells indicating that the requirement for CD4-dependent signaling events for IL-2 production is developmentally regulated and is particularly critical in previously activated effector T cells. In total, our results indicate that CD4 co-receptor dependent signaling events specifically regulate effector T cell survival and function. Further, these data suggest that CD4-dependent signaling events may protect against the decreased IL-2 production and resistance to cell death seen during chronic immune stimulation.     

A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

Background Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. Methods A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. Results By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. Conclusions The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease. P. Bühler and P. Wolf equally contributed to the work.