CD20-Specific Immunoligands Engaging NKG2D Enhance γδ T Cell-Mediated Lysis of Lymphoma Cells.

Human γδ T cells are innate-like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single-chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain-related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo-expanded γδ T cells against CD20-positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20-negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose-dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour-directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

CD137 (4-1BB) stimulation leads to metabolic and functional reprogramming of human monocytes/macrophages enhancing their tumoricidal activity.

Immunotherapies have heralded a new era in the cancer treatment. In addition to checkpoint inhibitors, agonistic antibodies against co-stimulatory immune receptors hold the potential to invoke efficient antitumor immunity. Targeting CD137 has gained momentum based on its ability to drive NK- and T-cell-based responses. CD137-engaging mAbs have already entered clinical trials for different types of tumors showing promising results. Despite the efforts to translate CD137-mediated immunotherapy into clinical practice, little remains known regarding the role of CD137 in human monocytes/macrophages.We found CD137 being expressed on monocytes of healthy controls and at even higher levels in patients with multiple myeloma or CLL. CD137HI(GH) monocytes displayed a distinct phenotypic, transcriptomic, and metabolic profile. They possessed an increased phagocytic capacity enabling superior antibody-dependent phagocytosis (ADPC) of multiple myeloma and lymphoma cells that were treated with anti-CD38 or anti-CD20 mAbs. Triggering CD137 promoted both metabolic and tumoricidal activity in an extracellular signal-regulated kinase (ERK)-dependent fashion. In addition, we observed a phenotypic, transcriptomic, and functional skewing towards a M1-like phenotype.Overall, we introduce CD137 as a positive immune checkpoint on human monocytes/macrophages, which can have therapeutic implications especially in view of synergistic effects when combining CD137 agonists with tumor-targeting antibodies.

A CD19-specific single-chain immunotoxin mediates potent apoptosis of B-lineage leukemic cells.

CD19 is a B-lineage-specific transmembrane signaling protein participating in the control of proliferation and differentiation. It is present at high surface density on chronic B-lymphocytic leukemia (B-CLL) cells and cells of other B-cell malignancies, and is a prime target for therapy with antibody-derived agents. Many attempts have been made to target malignant cells via CD19, but to date none of these agents have received drug approval. Here we report the design of a monovalent immunotoxin consisting of a CD19-specific single-chain Fv antibody fragment fused to a derivative of Pseudomonas Exotoxin A. This fusion protein induced efficient antigen-restricted apoptosis of several human leukemia- and lymphoma-derived cell lines including Nalm-6, which it eliminated at an effective concentration (EC(50)) of 2.5 nM. The agent displayed synergistic toxicity when used in combination with valproic acid and cyclosporin A in cell-culture assays. It induced apoptosis of primary malignant cells in 12/12 samples from B-CLL patients, including patients responding poorly to fludarabine, and of cells from one pediatric acute lymphoblastic leukemia patient. In NOD/SCID mice transplanted with Nalm-6 cells, the toxin prevented engraftment and significantly prolonged survival of treated mice. Owing to its efficient antigen-restricted antileukemic activity, the agent deserves further development towards clinical testing.

Luteolin protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway.

Oxidative stress is central to neuronal damage in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In consequence, activation of the cerebral oxidative stress defence is considered as a promising strategy of therapeutic intervention. Here we demonstrate that the flavone luteolin confers neuroprotection against oxidative stress via activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor central to the maintenance of the cellular redox homeostasis. Luteolin protects rat neural PC12 and glial C6 cells from N-methyl-4-phenyl-pyridinium (MPP+) induced toxicity in vitro and effectively activates Nrf2 as shown by ARE-reporter gene assays. This protection critically depends on the activation of Nrf2 since downregulation of Nrf2 by shRNA completely abrogates the protection of luteolin in vitro. Furthermore, the neuroprotective effect of luteolin is abolished by the inhibition of the luteolin-induced ERK1/2-activation. Our results highlight the relevance of Nrf2 for neural cell survival conferred by flavones. In particular, we identified luteolin as a promising lead for the search of orally available, blood brain barrier permeable compounds to support the therapy of neurodegenerative disorders.

An improved procedure for the generation of recombinant single-chain Fv antibody fragments reacting with human CD13 on intact cells.

A procedure was developed to generate recombinant single chain Fv (scFv) antibody fragments reacting with the extracellular domain of human cell surface antigen CD13 (hCD13; aminopeptidase N) on intact cells. Membrane fractions prepared from a stably transfected hCD13-positive murine NIH/3T3 cell line were used to immunize BALB/c mice, with the intention that hCD13 would be the major immunogenic molecule recognized by the immune system. Spleen RNA from the immunized mice served to generate a combinatorial scFv phage display library. The library was adsorbed against non-transfected NIH/3T3 or Sf21 insect cells to eliminate nonrelevant binders. The supernatant was then used for panning with either hCD13-transfected Sf21 insect cells or a hCD13-expressing human leukemia-derived cell line. Therefore, the key concepts of the procedure were the presentation of hCD13 as the sole human antigen on murine NIH/3T3 cells and a screening strategy where hCD13 was the major common antigen of the material used for immunization and panning. Two different hCD13-reactive phages were isolated and the soluble scFvs were expressed in E. coli and purified. The two scFvs, anti-hCD13-1 and anti-hCD13-3, differed at four amino acid positions in their V(H) regions and both had high affinities for hCD13 as determined by surface plasmon resonance (K(D)=7 and 33x10(-10) M, respectively). Both efficiently recognized hCD13 on intact cells. Therefore, the procedure allowed the production of high affinity scFvs reacting with a desired antigen in its native conformation without requiring extensive purification of the antigen and should be useful for the preparation of scFvs against other conformation-sensitive cell-surface antigens.

The novel immunotoxin HM1.24-ETA' induces apoptosis in multiple myeloma cells.

Despite new treatment modalities, the clinical outcome in a substantial number of patients with multiple myeloma (MM) has yet to be improved. Antibody-based targeted therapies for myeloma patients could make use of the HM1.24 antigen (CD317), a surface molecule overexpressed on malignant plasma cells and efficiently internalized. Here, a novel immunotoxin, HM1.24-ETA', is described. HM1.24-ETA' was generated by genetic fusion of a CD317-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA'). HM1.24-ETA' inhibited growth of interleukin 6 (IL-6)-dependent and -independent myeloma cell lines. Half-maximal growth inhibition was observed at concentrations as low as 0.3 nM. Target cell killing occurred via induction of apoptosis and was unaffected in co-culture experiments with bone marrow stromal cells. HM1.24-ETA' efficiently triggered apoptosis of freshly isolated/cryopreserved cells of patients with plasma cell leukemia and MM and was active in a preclinical severe combined immunodeficiency (SCID) mouse xenograft model. Importantly, HM1.24-ETA' was not cytotoxic against CD317-positive cells from healthy tissue (monocytes, human umbilical vein endothelial cells). These results indicate that CD317 may represent a promising target structure for specific and efficient immunotoxin therapy for patients with plasma cell tumors.

The novel tribody [(CD20)(2)xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells.

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)(2)xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development.

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.

Serum-free production and purification of chimeric IgA antibodies.

Natural IgA antibodies are abundantly produced in vivo to protect serosal surfaces from invading infectious organisms. However, the immunotherapeutic potential of IgA has hardly been explored, although there is evidence that recombinant IgA antibodies may broaden the armentarium to combat certain infectious or malignant diseases. One of the limitations for exploring IgA's therapeutic activity has been the difficulty to obtain enough recombinant material with desired specificity for in vivo studies. Here, we describe the production and purification of monomeric recombinant IgA1 and IgA2 antibodies under serum-free conditions. For antibody production, suspension adapted CHO-K1 cells and a glutamine synthetase selection vector were used, which resulted in specific production rates of up to 2.2 pg/cell/day. Purities of >95% of monomeric antibodies were obtained by a combination of affinity chromatography-using an anti-kappa-light chain matrix-and size exclusion chromatography. Purified antibodies displayed the expected biochemical characteristics and were functionally fully active. Importantly, all required reagents and methods are commercially available and not dependent on the specificity of the desired antibody. In addition, all employed technologies and methodologies are similar to those used for the production of therapeutic IgG antibodies - thus allowing further up-scaling and streamlining according to existing antibody production technologies. In conclusion, the described methodology may assist in the development of recombinant IgA antibodies for therapeutic applications.

Bispecific antibodies targeting cancer cells.

In recent years, antibody therapy has become a new treatment modality for tumour patients, although the majority of responses are only partial and not long lasting. Based on evidence that effector-cell-mediated mechanisms significantly contribute to antibody efficacy in vivo, several approaches are currently pursued to improve the interaction between Fc receptor-expressing effector cells and tumour target antigens. These approaches include application of Fc receptor-directed bispecific antibodies, which contain one specificity for a tumour-related antigen and another for a cytotoxic Fc receptor on immune effector cells. Thereby, bispecific antibodies selectively engage cytotoxic trigger molecules on killer cells, avoiding, for example, interaction with inhibitory Fc receptors. In vitro, chemically linked bispecific antibodies directed against the Fc gamma receptors Fc gamma RIII (CD16) and Fc gamma RI (CD64), and the Fc alpha receptor Fc alpha RI (CD89), were significantly more effective than conventional IgG antibodies. Recent animal studies confirmed the therapeutic potential of these constructs. However, results from clinical trials have been less promising so far and have revealed clear limitations of these molecules, such as short plasma half-lives compared with conventional antibodies. In this review, we briefly summarize the scientific background for bispecific antibodies, and describe the rationale for the generation of novel recombinant molecules. These constructs may allow us to more specifically tailor pharmacokinetic properties to the demands of clinical applications.