High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe.

Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon internalization into the acidic environment of endocytic organelles, whereas background fluorescence of noninternalized CypHer5E is minimal. The pH-dependency of the CypHer5E signal enables robust discrimination of antibody internalization from surface binding. The favorable signal-over-background ratio allows a homogeneous assay design with high-throughput fluorescence imaging in 384- and 1536-well formats. The biophysical readout of the primary internalization event substantially shortens incubation times compared to killing assays using toxin internalization. The assay was validated with tumor-relevant targets, including receptor tyrosine kinases (EGFR and HER2) and a class II cytokine receptor (TF) expressed by A431, AU565, and SKOV-3 cells and transient expression systems (CHO-S). Our method enables functional screening of large antibody libraries to identify therapeutic antibody candidates with internalization characteristics favorable for the development of ADCs.

Controlled Fab-arm exchange for the generation of stable bispecific IgG1.

The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2-3 d (including quality control).

High throughput screening for antibody induced complement-dependent cytotoxicity in early antibody discovery using homogeneous macroconfocal fluorescence imaging.

Complement-dependent cytotoxicity (CDC) represents an important Fc-mediated effector function of antibodies and is a quality often sought in candidates for therapeutic antibody development in cancer. Antibodies inducing potent CDC are relatively rare as the ability to induce CDC is strongly dependent on the antigen and epitope recognized as well as antibody isotype. To allow the identification of antibodies with optimal CDC characteristics in early stages of antibody discovery, we developed a homogeneous high throughput CDC assay, compatible with 384 and 1536 well formats and which therefore allows direct functional screening of very large panels of antibodies. Results obtained with our newly developed CDC method are consistent with those obtained with conventional assays. The assay proved to be robust, reliable over a wide reading window, easy to perform with low hands-on, high throughput, cost effective and applicable to crude hybridoma samples as typically available in early hybridoma discovery. In conclusion, we developed a novel high throughput assay for the identification of therapeutic antibody lead candidates with optimal CDC characteristics from large antibody libraries.

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.