Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system.

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

In vitro Fab display: a cell-free system for IgG discovery.

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system.

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.