Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

A novel human CD32 mAb blocks experimental immune haemolytic anaemia in FcgammaRIIA transgenic mice.

A fully human IgG1 kappa antibody (MDE-8) was generated, which recognised Fc-gamma receptor IIa (FcgammaRIIa) molecules on CD32 transfectants, peripheral blood monocytes, polymorphonuclear cells and platelets. This antibody blocked FcgammaRIIa ligand-binding via its F(ab')(2) fragment. Overnight incubation of monocytes with F(ab')(2) fragments of MDE-8 leads to a c. 60% decrease in cell surface expression of FcgammaRIIa. MDE-8 whole antibody induced a concomitant c. 30% decrease of FcgammaRI on THP-1 cells and monocytes. In humans FcgammaRIIa plays an important role in the clearance of antibody-coated red blood cells in vivo. As an equivalent of FcgammaRIIa does not exist in mice, the in vivo effect of MDE-8 was studied in an FcgammaRIIa transgenic mouse model. In these mice, antibody-induced anaemia could readily be blocked by MDE-8. These data document a new human antibody that effectively blocks FcgammaRIIa, induces modulation of both FcgammaRIIa and FcgammaRI from phagocytic cells, and ameliorates antibody-induced anaemia in vivo.

Flow cytometric determination of FcgammaRIIa (CD32) polymorphism.

A guanine to adenine point mutation results in an arginine (R) to histidine (H) substitution in FcgammaRIIa at residue 131 that strongly impacts receptor function. This FcgammaRIIa polymorphism is mostly typed by allele-specific polymerase chain reactions (PCR) or in functional assays, dependent on ligand binding. Both types of methods are laborious, time consuming, and not readily available in routine laboratories. We generated a panel of human antibodies against FcgammaRII, and one of them, MDE-9, selectively recognized the FcgammaRIIa-H131 allotype. MDE-9 was applicable to detect FcgammaRIIa-H131 in both flow cytometry and immunohistochemistry. MDE-9 was used to develop an FcgammaRIIa allotyping method based on flow cytometry. In a "single-tube assay", FITC-labeled MDE-9 (specific for FcgammaRIIa-H131) and Cy3-labeled mAb 41H16 (specific for FcgammaRIIa-R131) were added to 50 mul samples of whole blood. The results of flow cytometric FcgammaRIIa allotyping correlated completely with PCR genotyping. This novel allotyping assay should facilitate the screening of patients in a routine diagnostic setting. In addition, a combination of MDE-9 and 41H16 can be used in FcgammaRIIa-H/H131 homozygous individuals to detect FcgammaRIIa and FcgammaRIIb surface expression on monocytes. This is an important application of these antibodies because, to this day, no antibodies were available to specifically study the surface expression of FcgammaRIIb.

Dual mode of action of a human anti-epidermal growth factor receptor monoclonal antibody for cancer therapy.

Epidermal growth factor receptor (EGF-R) overexpression is common in a large number of solid tumors and represents a negative prognostic indicator. Overexpression of EGF-R is strongly tumor associated, and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1kappa) directed against EGF-R, developed using human Ig transgenic mice. mAb 2F8 effectively blocked binding of EGF and TGF-alpha to the EGF-R. At saturating concentrations, 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concentrations, associated with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo analysis of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor saturation and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we observed potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.

Resolution of psoriasis upon blockade of IL-15 biological activity in a xenograft mouse model.

Psoriasis is a chronic inflammatory disease of the skin characterized by epidermal hyperplasia, dermal angiogenesis, infiltration of activated T cells, and increased cytokine levels. One of these cytokines, IL-15, triggers inflammatory cell recruitment, angiogenesis, and production of other inflammatory cytokines, including IFN-gamma, TNF-alpha, and IL-17, which are all upregulated in psoriatic lesions. To investigate the role of IL-15 in psoriasis, we generated mAb's using human immunoglobulin-transgenic mice. One of the IL-15-specific antibodies we generated, 146B7, did not compete with IL-15 for binding to its receptor but potently interfered with the assembly of the IL-15 receptor alpha, beta, gamma complex. This antibody effectively blocked IL-15-induced T cell proliferation and monocyte TNF-alpha release in vitro. In a human psoriasis xenograft model, antibody 146B7 reduced the severity of psoriasis, as measured by epidermal thickness, grade of parakeratosis, and numbers of inflammatory cells and cycling keratinocytes. These results obtained with this IL-15-specific mAb support an important role for IL-15 in the pathogenesis of psoriasis.

Experimental autoimmune myasthenia gravis in mice expressing human immunoglobulin loci.

Antibodies (Abs) specifically directed against the muscular acetylcholine receptor (AChR) mediate the pathogenesis of myasthenia gravis (MG). The animal model experimental autoimmune MG (EAMG) can be induced by passive transfer or by active immunization of anti-AChR Abs. We report a new EAMG mouse model that generates human anti-AChR Abs upon immunization with Torpedo AChR (tAChR). Mice transgenic for human mu, gamma1, and kappa germ line genes (HuMAb-Mice) were immunized with tAChR. Serum titers of anti-tAChR Abs were in the nanomolar range, and anti-rodent AChR Abs were in picomolar range. Some HuMAb-Mice had signs of muscle weakness, clearly indicating their susceptibility to EAMG. Human Ab-mouse AChR complexes were found at the neuromuscular junction, while AChR loss was up to 65%. Spleen and lymph nodes were used for producing hybridomas. Of the anti-tAChR monoclonal Ab-producing hybridomas, 2% had cross-reactivity with rodent AChR and none with human AChR. Immunization with a fusion protein, Trx-Halpha1-210, displaying the human main immunogenic region did not result in EAMG or the generation of human anti-human AChR monoclonal Abs. These experiments show that the HuMAb-Mouse represents a suitable model to generate and study the effects of human anti-AChR Abs in vivo.

Interferon-gamma:interleukin 4 ratios and associated type 1 cytokine expression in juvenile rheumatoid arthritis synovial tissue.

OBJECTIVE: To compare synovial tissue cytokine mRNA expression between patients with juvenile rheumatoid arthritis (JRA) and a heterogeneous group of non-autoimmune arthropathies (controls) with respect to type 1/type 2 balance. METHODS: Thirty-five JRA (average 9.1 years' disease duration) and 13 control synovial tissues were studied. As a measure of the type 1/type 2 cytokine balance in a subset of the JRA and control tissues, interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) mRNA levels were measured by competitive fragment reverse transcription-polymerase chain reaction. To quantitate additional cytokines relevant to this balance, multiprobe ribonuclease protection assays were employed measuring IL-5, IL-10, IL-13, IL-15, IL-18, and IL-12 (p35 and p40 subunits). Immunohistochemistry was performed on JRA tissues using antibodies specific for IL-15 and IL-18. RESULTS: A higher IFN-gamma:IL-4 ratio (p = 0.034) was found in JRA tissues compared to controls. JRA tissues also displayed higher mRNA levels of IL-12p35 (p = 0.021), IL-15 (p = 0.002), and IL-18 (p = 0.017), but not IL-4 and IL-10. IFN-gamma expression in JRA, but not controls, correlated strongly with IL-12p35 (r = 0.63) and IL-12p40 (r = 0.73) levels. A subset of IL-15+ and IL-18+ cells was detected in JRA synovial tissues, largely within perivascular aggregates. CONCLUSION: JRA synovial tissue cytokine expression patterns indicate a type 1 bias, even in the later stages of disease. The strong correlation between IFN-gamma and IL-12 in JRA suggests a prominent role for IL-12 in promoting the type I bias, while IL-15 and IL-18 may also indirectly increase IFN-gamma expression and further bias the immune response.