Gundram Jung

Interaction of Monocytes with NK Cells upon Toll-Like Receptor-Induced Expression of the NKG2D Ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1–3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-γ production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-γ may communicate directly during an innate immune response to infections in humans.

Cutting Edge: Predetermined Avidity of Human CD8 T Cells Expanded on Calibrated MHC/Anti-CD28-Coated Microspheres

Cytotoxic CD8 T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial APCs (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. We show that, by controlling the MHC density on aAPCs, high- or low-avidity tumor-directed human CTL lines can be raised effectively in vitro if costimulation via CD28 and IL-12 is provided. Compared with low-avidity CTL lines, high-avidity CTLs need 100- to 1000-fold less peptide for activation, bind more MHC tetramers, and, as expected, are superior in recognizing tumor cell lines expressing Ag. We believe that the possibility to raise Ag-specific T cells with predetermined avidity will be crucial for the future use of aAPCs in immunotherapeutical settings.

Local immunotherapy of glioma patients with a combination of 2 bispecific antibody fragments and resting autologous lymphocytes : Evidence for in situ T-cell activation and therapeutic efficacy

After adoptive transfer of pre‐activated lymphocytes into the operation cavity of glioma patients, tumor regression and improved survival have been reported in some patients. Results were most impressive when bispecific antibodies with tumor × CD3 specificity were also applied. In this study, we attempted to avoid time‐consuming pre‐activation procedures for adoptively transferred cells by using a combination of bispecific antibodies directed to the EGF receptor (EGFR) on tumor cells and to CD3 and CD28 on T cells. Eleven patients with high‐grade malignant glioma received 3 injections of 2 bispecific antibody fragments (EGFR × CD3 and EGFR × CD28) together with freshly isolated autologous lymphocytes via an Ommaya reservoir. Intracavitary fluid aspirated during immunotherapy was examined for markers of T‐cell activation. Increased levels of soluble IL‐2 receptor and TNF‐α were detected in the intracavitary fluid of all patients tested. Two of the 11 treated patients experienced a beneficial response to therapy as defined by a transient contrast enhancement in subsequent MRI scans and prolonged survival. Side effects were transient and consisted of fever, nausea, headache and aggravation of pre‐existing neurologic deficits. These adverse effects were most likely due to the antibody construct containing anti‐CD3 specificity. Two patients developed cerebral edema and required steroid treatment.

Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells

The in vitro generation of cytotoxic T lymphocytes (CTLs) for anticancer immunotherapy is a promising approach to take patient-specific therapy from the bench to the bedside. Two criteria must be met by protocols for the expansion of CTLs: high yield of functional cells and suitability for good manufacturing practice (GMP). The antigen presenting cells (APCs) used to expand the CTLs are the key to achieving both targets but they pose a challenge: Unspecific stimulation is not feasible because only memory T cells are expanded and not rare naïve CTL precursors; in addition, antigen-specific stimulation by cell-based APCs is cumbersome and problematic in a clinical setting. However, synthetic artificial APCs which can be loaded reproducibly with MHC-peptide monomers and antibodies specific for costimulatory molecules could resolve these problems. The purpose of this study was to investigate the potential of complex synthetic artificial APCs in triggering the costimulatory molecules CD28 and 4-1BB on the T cell. Anti-4-1BB antibodies were added to an established system of microbeads coated with MHC-peptide monomers and anti-CD28. Triggering via CD28 and 4-1BB resulted in strong costimulatory synergy. The quantitative ratio between these signals determined the outcome of the stimulation with optimal results when anti-4-1BB and anti-CD28 were applied in a 3:1 ratio. Functional CTLs of an effector memory subtype (CD45RA− CCR7−) were generated in high numbers. We present a highly defined APC platform using off-the-shelf reagents for the convenient generation of large numbers of antigen-specific CTLs.

RANKL Expression, Function, and Therapeutic Targeting in Multiple Myeloma and Chronic Lymphocytic Leukemia

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Role of target antigen in bispecific-antibody-mediated killing of human glioblastoma cells : A pre-clinical study

Bispecific antibodies (bsAbs) directed to tumor‐associated antigens and to receptors mediating T‐cell activation, such as the TCR/CD3 complex and the co‐stimulatory CD28 molecule, are capable of activating T cells at the surface of tumor cells, resulting in tumor‐cell killing. Here we report the pre‐clinical characterization of bispecific‐antibody fragments (bsFab2) directed to 2 different glioblastoma‐associated antigens: the EGF receptor (EGFR) and a chondroitin‐sulfate proteoglycan (CSPG). Using cultured glioblastoma cells expressing both target antigens, we found that the ability of anti‐tumor × anti‐CD28 bsFab2 to mediate “targeted T‐cell co‐stimulation” is superior for constructs targeting the CSPG molecule, correlating with an approximately 6‐fold higher expression level of this antigen on the cell surface. In contrast, bsFab2 triggering CD3 are more effective if they contain EGFR‐target specificity. This indicates that the activity of anti‐tumor × anti‐CD3 constructs critically depends on properties of the antigen other than its expression level on the cell surface, e.g., its mobility in the membrane. These findings prompted us to use EGFR‐targeting bsFab2 in an ongoing clinical trial with glioma patients. Int. J. Cancer 80:612–616, 1999.

Bispecific antibody fragments with CD20 × CD28 specificity allow effective autologous and allogeneic T-cell activation against malignant cells in peripheral blood and bone marrow cultures from patients with B-cell lineage leukemia and lymphoma

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation, such as the TCR/CD3 complex and the costimulatory receptor CD28, are capable of mediating T-cell activation resulting in tumor cell killing. In this study, we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2, when used alone rather than in combination, markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore, these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and, in particular, of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients.

A flow cytometric long-term cytotoxicity assay

A method to evaluate cytotoxic effects applicable over a wide range of incubation times has been developed. It is based on quantification by flow cytometry of dead and viable target cells stained by covalently binding the fluorescent dye fluorescein isothiocyanate (FITC). The staining with FITC did not affect cell viability and growth parameters and was stable enough to identify target cells for at least 2 days. The flow cytometric analysis of the cell mixture was performed in a test system with activated CD8+ lymphocytes as effector cells and melanoma M21 cells as targets in the presence of appropriate bispecific antibodies and revealed a rather complex pattern composed of several distinct cell subsets which were identified by use of antibodies to lymphocyte antigens. The assay compared favourably with results from a conventional 51Cr release assay obtained after 4 h and 8 h of incubation and from a target cell adherence assay obtained after 24 h of incubation. The application of the method described herein is especially advantageous for the evaluation of long-term cytotoxic effects. Furthermore, it provides valuable multi-parameter information which is useful for elucidating mechanisms of cytotoxicity.

Supraagonistic, bispecific single-chain antibody purified from the serum of cloned, transgenic cows induces T-cell-mediated killing of glioblastoma cells in vitro and in vivo

Here we characterize the antitumor activity of a recombinant bispecific single‐chain antibody isolated from the serum of cloned transgenic cows. The antibody, termed r28M, is directed to a melanoma‐associated proteoglycan, also expressed on glioblastoma cells, and to human CD28. Bound to tumor cells, r28M induced exceedingly efficient supraagonistic T‐cell activation via the CD28 molecule without an additional stimulus via the TCR/CD3 complex. In vitro, T cells and NK cells contributed to tumor cell killing after r28M‐mediated activation of peripheral blood mononuclear cells. However, NK activity depended on T‐cell‐derived cytokines. In vivo, r28M markedly inhibited the growth of human glioblastoma cells in nude mice. The serum half‐life of the protein after i.v. injection was approximately 6 hr. Thus, r28M is unique not only in inducing supraagonistic CD28‐mediated T‐cell activation against tumor cells in vitro and in vivo, it also meets 2 additional requirements that are critical for clinical application: a relatively long serum half‐life and the possibility of obtaining large amounts of active material from cloned transgenic livestock.

An Fc‐optimized NKG2D‐immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia

Recruitment of Fc‐receptor‐bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibodys Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor‐associated expression of NKG2DL for targeting leukemic cells by NKG2D–immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D–Fc containing a wild‐type Fc part (NKG2D–Fc–WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D–Fc–ADCC) and abrogated (NKG2D–Fc–KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D–Fc–KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D–NKG2DL interaction. NKG2D–Fc–WT already enhanced antileukemia reactivity by inducing antibody‐dependent cellular cytotoxicity (ADCC) with NKG2D–Fc–ADCC mediating significantly stronger effects. Parallel application of NKG2D–Fc–ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor‐associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D–Fc–ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.