Generating a T cell tumor-specific immune response in vivo: can flt3-ligand-generated dendritic cells tip the balance?

flt3 ligand (FL) is a growth factor that induces hematopoietic progenitor cell and dendritic cell (DC) expansion when administered to mice. Lymphoid-related (CD8α⁺) and myeloid-related (CD8α⁻) DC are transiently expanded in multiple tissues. Treatment of tumor-bearing mice with FL results in slower tumor growth and, in some cases, tumor rejection and the development of tumor-specific T cell immunity. The clinical use of DC as cellular vehicles for tumor antigen presentation to generate a tumor-specific T cell response is under investigation. DC are currently generated ex vivo, pulsed with antigen, and then infused into patients, and much effort is being directed toward optimizing each of these steps. Administration of FL to humans induces a profound increase in circulating DC. The availability of a large number of DC generated in vivo has important implications for tumor immunotherapy approaches. Received: 13 May 1999 / Accepted: 14 June 1999     

2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G_D2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25^hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.     

High-scale expansion of melanoma-reactive TIL by a polyclonal stimulus: predictability and relation with disease advancement

 The rationale of treating melanoma patients by infusion with tumor-infiltrating leukocytes (TIL) is to perform an adoptive therapy through injection of tumor-specific T cells. Nonetheless, methods currently used for ex vivo TIL expansion have not been evaluated for their efficacy to expand TAA-specific T cells. We have addressed this question here, using a culture method in which high TIL growth was induced by a polyclonal T cell stimulus. Intracellular cytokine assays were performed to measure the proportion of T cells responding to autologous tumor cells among the lymphocytes from lymph node biopsies (TIL) of 26 patients with stage III melanoma. The data show that TIL from 18 of these patients contained detectable amounts of tumor-specific T cells before expansion. Although they decreased somewhat in percent abundance during expansion, they were still present afterwards, ranging from 0.3 to 13.8%. Since a median number of 1.7 × 10¹⁰ TIL was obtained from these patients (starting from 3.6 × 10⁶ TIL), a total amount of tumor-reactive cytokine-secreting TIL of between 2.8 × 10⁶ and 1.12 × 10⁹ was obtained in each case from 18 patients. The TIL populations from 8 patients did not contain tumor-reactive T cells: neither before expansion, nor after expansion. Lack of tumor-reactive TIL only occurs for patients bearing several tumor-invaded lymph nodes (40%), but not for those having a single invaded lymph node. Therefore, high numbers of tumor-reactive T cells can be produced, through a polyclonal TIL stimulation, from most early stage III melanoma patients but from only about half of the patients with a more disseminated disease. For this last group, the possibility of getting tumor-reactive TIL can be predicted by checking the presence of these cells before expansion. Received: 16 November 2000 / Accepted: 18 January 2001     

Bispecific antibody treatment of murine B cell lymphoma

 This report summarizes our experimental data concerning the use of bispecific antibodies (bsAb) for the treatment of the murine BCL1 B cell lymphoma model. Initially we used a hybrid-hybridoma-derived bsAb with specificity for the TcR/CD3 complex on T cells and the idiotype of the membrane-bound IgM on the tumor cells. The bsAb used as a single agent could cure animals with a low tumor load (resembling minimal residual disease). However, in experiments aimed at increasing the therapeutic effect in animals with a higher tumor burden, we could demonstrate the importance of additional T-cell-costimulatory signals and the careful timing of the bsAb administration. Recently we have generated a bispecific single-chain Fv (bsscFv) fusion protein with the same dual specificity as the hybrid-hybridoma-derived bsAb. Immunotherapy with this smaller molecule also resulted in tumor elimination in BCL1-bearing mice. A second bsscFv (α-CDl9×α-CD3) with a broader applicability is now being characterized and tested in vivo. Accepted: 14 October 1997     

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon α and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3⁺CD56⁺ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis^y antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000     

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct

BscCD19xCD3 is a bispecific single-chain antibody construct with exceptional cytotoxic potency in vitro and in vivo. Here, we have investigated the biological activity of bscCD19xCD3 in chimpanzee, the only animal species identified in which bscCD19xCD3 showed bispecific binding, redirected B-cell lysis and cytokine production comparable to human cells. Pharmacokinetic analysis following 2-h intravenous infusion of 0.06, 0.1 or 0.12 μg/kg of bscCD19xCD3 as part of a dose escalation study in a single female chimpanzee revealed a half-life of approximately 2 h and elimination of the bispecific antibody from circulation within approximately 8 h after the end of infusion. This short exposure to bscCD19xCD3 elicited a transient increase in serum levels of IFNγ, IL-6, IL-2, soluble CD25, and transiently upregulated expression of CD69 and MHC class II on CD8-positive cells. Cytokine release and upregulation of T-cell activation markers were not observed with vehicle controls. A multiple-dose study using 5 weekly doses of 0.1 μg/kg in two animals also showed transient cytokine release and an activation of peripheral T cells with a first-dose effect, accompanied by a transient lymphopenia. While oscillations of T-cell counts were relatively even during repeated treatments, the amplitudes of peripheral B cells declined with every infusion, which was not observed in a vehicle control animal. Our data show that bscCD19xCD3 can be safely administered to chimpanzees at dose levels that cause fully reversible T-cell activation and, despite a very short exposure time, cumulative loss of peripheral B lymphocytes. A clinical trial testing prolonged administration of bscCD19xCD3 (MT103) for improving efficacy is currently ongoing.     

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4⁺ and CD8⁺ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4⁺ and CD8⁺ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4⁺ and CD8⁺ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4⁺ and CD8⁺ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4⁺ and CD8⁺ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2). Received: 12 November 1999 / Accepted: 2 January 2000     

Increased potency of Fc-receptor-targeted antigens

 A major challenge for using native and modified T cell epitopes to induce or suppress immunity relates to achieving efficient uptake and processing by antigen-presenting cells (APC) in vivo. IgG Fc receptors, which are expressed constitutively by professional APC including monocytes and dendritic cells, have long been known to mediate antigen uptake in a manner leading to efficient T cell activation. We have previously demonstrated enhanced presentation of antigenic and antagonistic peptides by targeting them to the type I Fc receptor for IgG (FcγRI, CD64) on human monocytes. In the present report we review the literature suggesting that CD64-targeted antigens are likely to be effective in vivo, and present data demonstrating enhanced immunogenicity in CD64 transgenic mice of a fusion protein that combines the specificities of HIV gp120 and the humanized anti-CD64 monoclonal antibody H22. Overall, these studies suggest that targeting antigens to CD64 represents an effective approach to enhancing the effectiveness of vaccines in vivo. Accepted: 14 October 1997     

Moderate and exhaustive endurance exercise influences the interferon-γ levels in whole-blood culture supernatants

The aim of this study was to investigate whether moderate or exhaustive endurance exercise influences cytokine levels in whole-blood culture supernatants after stimulation. Therefore, eight healthy subjects were first exposed to moderate exercise on a cycle ergometer for 30 min at 70% of their 4-mmol/l lactic acid (anaerobic) threshold, and 1 week later to exhaustion (for 90 min) at their anaerobic threshold. Blood samples were taken before, 30 min after and 24 h after each exercise bout. The following lymphocyte subpopulations were determined: CD14-positive(+)/CD45+, CD4+, CD8+, and CD16+. Cytokine levels in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Production of interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α were induced with lipopolysaccharides (LPS), and that of IL-2 and interferon (IFN)-γ with staphylococcal enterotoxin B (SEB) and phytohaemagglutinin (PHA). Cortisol levels were also determined by ELISA. The lymphocyte subset distribution was observed to be unchanged after moderate exercise. Thirty minutes after exhaustive exercise, the CD16+ count was found to be significantly lower, whereas 24 h later the CD4+ count was significantly higher than pre-exercise counts. Moderate exercise influenced the IFN-γ production (PHA-stimulated), which increased significantly from 974 (391) pg/ml before exercise to 1450 (498) pg/ml 24 h later. Thirty minutes after exhaustive exercise the IFN-γ level in the supernatants (SEB-stimulated) was significantly decreased (from 14470 (11840) pg/ml before exercise to 6000 (4950) pg/ml after exercise). The IL-1β and TNF-α production per monocyte was also significantly reduced. Accepted: 19 February 1997     

Adoptive-transfer therapy of tumors with the tumor-specific primary cytotoxic T cells induced in vitro with the B7.1-transduced MCA205 cell line

We show that the tumor-specific primary cytotoxic T lymphocytes (CTL) induced in vitro with the MCA205 fibrosarcoma cells transduced with the B7.1 (CD80) gene are highly effective in adoptive-transfer therapy of the parental tumors. The MCA205 fibrosarcoma cell line was transduced with the retroviral vectors encoding the B7.1 gene and tested for their efficiency as stimulators in short-term (5 days) mixed lymphocyte/tumor cell cultures with highly purified syngenic, unprimed T cells as responders. The induction of the CTL required the presence of a low dose of interleukin-2 (25 U/ml). The injection of the CTL prevented colony formation by the intravenously injected tumor cells in a lung colonization assay in which the CTL were injected after inoculation of tumor cells. We also showed that the adoptive transfer of the same T cells was effective in delaying the growth of the subcutaneously injected tumor cells. These results imply that the short-term mixed lymphocyte/tumor cell culture with the tumor cells transduced with the gene for the B7.1 costimulatory molecule is potentially a good source of CTL for adoptive-transfer therapy of tumors. Received: 30 June 1998 / Accepted: 5 August 1998     

Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer

 Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL_17-1A-VH_17-1A-VH_CD3-VL_CD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (⁵¹Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8⁺ as well as CD4⁺ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8⁺ cells induced high ⁵¹Cr release within 4 h while CD4⁺ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto. Accepted: 14 October 1997     

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K _D = [1.7 ± 0.2] × 10⁻¹⁰ M) and IgG3 (K _D = [1.1 ± 0.2] × 10⁻¹⁰ M) were similar to those of glycosylated rhFcγRI.     

A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells

The single-chain triplebody HLA-ds16-hu19 consists of three single-chain Fv (scFv) antibody fragments connected in a single polypeptide chain. This protein with dual-targeting capacity mediated preferential lysis of antigen double-positive (dp) over single-positive (sp) leukemic cells by recruitment of natural killer (NK) cells as effectors. The two distal scFv modules were specific for the histocompatibility protein HLA-DR and the lymphoid antigen CD19, the central one for the Fc gamma receptor CD16. In antibody-dependent cellular cytotoxicity (ADCC) experiments with a mixture of leukemic target cells comprising both HLA-DR sp HuT-78 or Kasumi-1 cells and (HLA-DR plus CD19) dp SEM cells, the triplebody mediated preferential lysis of the dp cells even when the sp cells were present in ≤20-fold numerical excess. The triplebody promoted equal lysis of SEM cells at 2.5-fold and 19.5-fold lower concentrations than the parental antibodies specific for HLA-DR and CD19, respectively. Finally, the triplebody also eliminated primary leukemic cells at lower concentrations than an equimolar mixture of bispecific single-chain Fv fragments (bsscFvs) separately addressing each target antigen (hu19-ds16 and HLA-ds16). The increased selectivity of targeting and the preferential lysis of dp over sp cells achieved by dual-targeting open attractive new perspectives for the use of dual-targeting agents in cancer therapy.     

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.     

Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco.

An anti-gibberellin A24/19 single-chain Fv gene was constructed from gamma and kappa genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se. The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed. When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6% of the total soluble protein. On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all. The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1.     

Induction of H2-Uptake and Nitrogenase Activities in the Cyanobacterium Anabaena variabilis ATCC 29413: Effects of Hydrogen and Organic Substrate

A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H₂-uptake [2–7 μmoles of H₂ · mg⁻¹ (Chl a) · h⁻¹] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H₂ on in vivo H₂-uptake was observed during the early stages of nitrogenase induction. However, exogenous H₂ does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth. Received: 22 November 1995 / Accepted: 22 December 1995     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

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.     

Effect of Dietary Selenium and Cancer Cell Xenograft on Peripheral T and B Lymphocytes in Adult Nude Mice

Selenium (Se) is known to regulate tumorigenesis and immunity at the nutritional and supranutritional levels. Because the immune system provides critical defenses against cancer and the athymic, immune-deficient NU/J nude mice are known to gradually develop CD8⁺ and CD4⁺ T cells, we investigated whether B and T cell maturation could be modulated by dietary Se and by tumorigenesis in nude mice. Fifteen homozygous nude mice were fed a Se-deficient, Torula yeast basal diet alone (Se−) or supplemented with 0.15 (Se+) or 1.0 (Se++) mg Se/kg (as Na₂SeO₄) for 6 months, followed by a 7-week time course of PC-3 prostate cancer cell xenograft (2 × 10⁶ cells/site, 2 sites/mouse). Here, we show that peripheral B cell levels decreased in nude mice fed the Se −  or Se++ diet and the CD4⁺ T cell levels increased in mice fed the Se++ diet. During the PC-3 cell tumorigenesis, dietary Se status did not affect peripheral CD4⁺ or CD8⁺ T cells in nude mice whereas mice fed with the Se++ diet appeared to exhibit greater peripheral CD25⁺CD4⁺ T cells on day 9. Dietary Se status did not affect spleen weight in nude mice 7 weeks after the xenograft. Spleen weight was associated with frequency of peripheral CD4⁺, but not CD8⁺ T cells. Taken together, dietary Se at the nutritional and supranutritional levels regulates peripheral B and T cells in adult nude mice before and after xenograft with PC-3 prostate cancer cells.     

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998