ANTIBODY ENGINEERING ON THE SURFACE OF CHO CELLS

摘要

From discovery to market, antibody therapeutics are developed using several expression platforms. Antibodies are often discovered from isolated mouse or human B-cells, engineered for affinity and specificity on the surface of phage, bacteria, or yeast, and manufactured in FDA-approved mammalian cell lines. This functional engineering detour in non-mammalian platforms introduces the opportunity to adversely impact expression and stability of the final product in the mammalian production cell line. To address this shortcoming, a CHO cell display platform was developed in which the antibody therapeutic can be engineered in the CHO manufacturing cell line, streamlining the process (Fig.1). CHO cell display of an antibody Fab fragment was developed and optimized using a semi-stable episomal system capable of maintenance of a transfected plasmid for several weeks, which allows multiple rounds of flow cytometric sorting and regrowth of promising clones. To test the utility of the CHO surface display platform and screening process, a randomly mutated library was made from a low affinity antibody parent and screened for improved binding. The target antigen for this test case was HER2, a well-characterized receptor over-expressed in approximately 20% of breast cancer cases. The anti-HER2 library was subjected to two rounds of flow cytometric screening for improved antigen binding while controlling for expression level. This resulted in nine unique clones, of which six exhibited improved HER2 binding relative to the parent upon subsequent analysis of individual clones in the displayed format. Seven of the nine sequences were sub-cloned for CHO cell expression as full-length, soluble antibodies and characterized for expression level, affinity, and stability. All seven full-length antibodies were expressed at levels and with stabilities comparable to the parent, and several had improved binding affinity relative to the parent. Although the anti-HER2 antibody 4D5 (Herceptin) has been an effective breast cancer therapeutic, it exhibits undesired binding to myocardiocytes, which can result in adverse reactions to therapy. The low pH tumor microenvironment provides an opportunity to engineer the antibody to selectively bind HER2 at low pH (-6) and avoid binding non-target cells residing in the neutral (~pH 7.4) normal tissue microenvironment. A library of4D5 Fab variants was displayed on CHO cells and screened for preferential binding to HER2 under low pH conditions. Data on clonal expression and pH-dependent ligand binding will be shown. In both the CHO surface display proof-of-principle affinity maturation and low pH selective binding screen presented, the antibodies were improved in a commonly used manufacturing cell line, which is expected to limit the need for further expression optimization. The CHO surface display technology and techniques represent a generalizable method for the streamlined development of therapeutic antibodies.