Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics.

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format. Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.

Antibody Selection on FFPE Tissue Slides.

Standard antibody phage-display panning uses purified proteins, antigen-transfected cells, or tumor cell lines as target structure to generate specific antibodies. Here, we describe a method for the selection of specific antibodies by phage panning against routine formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides. Selected antibody fragments recognize disease-associated antigens in its native conformation, suitable for the development of targeted diagnostic and therapeutic agents.

Development of a monoclonal sandwich ELISA for direct detection of bluetongue virus 8 in infected animals.

Bluetongue is an infectious viral disease which can cause mortality in affected ruminants, and tremendous economic damage via impacts upon fertility, milk production and the quality of wool. The disease is caused by bluetongue virus (BTV) which is transmitted by species of Culicoides biting midge. Rapid detection of BTV is required to contain disease outbreaks and reduce economic losses. The purpose of this study was to develop a monoclonal sandwich ELISA for direct detection of BTV in infected animals. Phage display technology was used to isolate BTV specific antibody fragments by applying the human scFv Tomlinson antibody libraries directly on purified BTV-8 particles. Three unique BTV-8 specific human antibody fragments were isolated which were able to detect purified BTV particles and also BTV in serum of an infected sheep. A combination of a human/mouse scFv-Fc chimeric fusion protein and a human Fab fragment in a sandwich ELISA format was able to detect BTV specifically with a limit of detection (LOD) of 104 infectious virus particles, as determined by tissue culture titration. This approach provided pilot data towards the development of a novel diagnostic test that might be used for direct detection of BTV-8 particles.

Phage display-based on-slide selection of tumor-specific antibodies on formalin-fixed paraffin-embedded human tissue biopsies.

Phage display is an effective method for the generation of target-specific human antibodies. Standard phage display panning use purified proteins, antigen-transfected cells or tumor cell lines as target structure to generate specific antibodies. However, recombinant proteins can be difficult to express and purify in their native conformation and suitable cell lines are not always available. Additionally the antigen expression profile may change during cultivation and thus differ from the malignant cells in patient. Here we describe a method for the selection of specific antibodies from phage display libraries by panning against formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides, using small cell lung cancer (SCLC) as a case study. The human Tomlinson single-chain variable fragment (scFv) phage libraries I and J were panned against SCLC FFPE tissue slides for positive selection and healthy lung tissue for subtraction. The specificity of the selected scFv antibodies was confirmed in vitro by ELISA on immobilized SCLC cell membranes, by flow cytometry using the SCLC cell lines NCI-H69, NCI-H82 and DMS 273, and ex vivo against tissue microarrays containing 35 different SCLC samples and 20 types of normal organs. We monitored the internalization of three selected scFv antibodies and fused them with Pseudomonas exotoxin A (ETA') to produce immunotoxins whose cytotoxicity was confirmed by cell viability and apoptosis assays on different SCLC cell lines, achieving IC50 values of up to 23nM. The selection of SCLC-specific scFv antibodies by panning against FFPE tissue slides circumvents the challenges of using purified antigens or cell lines for antibody selection.

Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia.

The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off­target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2­derived Kasumi­1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AML­selective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA'). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients.

In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells.

PURPOSE: Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA' against EGFR(+) RMS cells in vitro and ex vivo. METHODS: We tested the specific binding and internalization behavior of 425(scFv)-ETA' in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA' was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients. RESULTS: We confirmed the specific binding of 425(scFv)-ETA' to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR(+) RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line. CONCLUSIONS: This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA'-based IT on RMS cells. We confirmed the selective killing with IC50 values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material.

CD30 as a therapeutic target for lymphoma.

Hodgkin's lymphoma (HL) and ALK(+) anaplastic large-cell lymphoma (ALCL) have become highly curable due to the success of modern regimens of chemotherapy and radiotherapy. However, up to one-third of the patients experience relapse or do not respond to first-line therapy, and half of them relapse again after secondary therapy with limited options for further treatment. In the last 15 years, monoclonal antibodies (mAbs) directed to surface receptors became a new and valuable therapeutic option in many hematologic malignancies. Due to its restricted expression on normal activated lymphocytes and its high expression on malignant cells, CD30 represents an attractive target molecule for HL and ALCL therapy. However, unconjugated CD30 mAbs have demonstrated a lack of objective clinical responses in patients with recurrent HL. CD30 exhibits complex signaling pathways, and binding of its natural ligand or anti-CD30 mAbs can induce apoptosis but may also promote proliferation and activation depending on the cellular context. Moreover, CD30 rapidly internalizes after crosslinking, which counteracts efficient recruitment of immunologic effectors but also provides the opportunity to transfer cytotoxic payloads coupled to CD30-specific mAbs into the tumor cells. Several tumor targeting approaches have been studied, including radio-immunoconjugates, immunotoxins, immunoRNases, immunokinases, and antibody drug conjugates (ADCs). In 2011, the ADC brentuximab-vedotin, consisting of the CD30-specific chimeric mAb cAC10 and the potent tubulin toxin monomethyl auristatin E, gained regulatory approval as a well tolerated and highly active drug in patients with refractory and relapsed HL and ALCL. SGN-35 is on the way to being incorporated in the standard management of CD30(+) lymphoma with significant therapeutic impact. This review gives a critical overview about anti-CD30 therapies with unconjugated, engineered, and conjugated mAbs and the therapeutic challenges of treatment of CD30(+) lymphoma.