Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Xu, Yiren; Lee, John; Tran, Cuong; Heibeck, Tyler H.; Wang, Willie D.; Yang, Jun; Stafford, Ryan L.; Steiner, Alexander; Sato, Aaron K.; Hallam, Trevor J.; Yin, Gang

doi:10.4161/19420862.2015.989013

Cited by 71 publications

(64 citation statements)

References 66 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Pharmacokinetics studies in rats showed that BEAT antibodies benefited from a prolonged half-life similar to that of previously reported Hc heterodimers (26, 27); for example, an elimination half-life ( t ½ ) of 164 h following i.v. injection of 10 mg/kg of BEAT 2/3 was observed (supplemental Table S2; note that antibody U1-59 has been reported to cross-react with rat HER3 (22)).…”

Section: Resultssupporting

confidence: 79%

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Skegro

Stutz

Ollier

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/β, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/β constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported.

show abstract

Section: Resultssupporting

confidence: 79%

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Skegro

Stutz

Ollier

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…As reconstitution efficiency is highly dependent on antibody fragment concentration and the molar ratio of the two monovalent antibody fragments, it has been reported that purification of product-related impurities is very difficult and remains a manufacturing challenge, especially in small-scale screening applications. [14,36] The method presented herein allows for a one-step purification without elution of the desired antibody due to a His-Tag fusion to each antibody fragment as well as the resulting intein side product. Non-reconstituted antibody fragments and processed intein can be extracted via Ni 2+ beads in 384well format.…”

Section: Discussionmentioning

confidence: 99%

Intein mediated high throughput screening for bispecific antibodies

Hofmann

Schmidt

Ciesielski

et al. 2020

mAbs

View full text Add to dashboard Cite

Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.

show abstract

“…To promote heterodimerization of two heavy chains with different specificities, CH3 domains are engineered to bear either a "knob" or a "hole" in each antibody heavy chain by amino acid replacement [122]. Recently, this technology has been applied in an E. coli-based cell-free translation system creating bispecific antibodies in different designs (e.g., two-armed heterodimeric scFv-knobs-into-holes; one-armed assymetric BiTE knobs-into-holes with tandem scFv) [123].…”

Section: Discussionmentioning

confidence: 99%

Cell-Free Synthesis Meets Antibody Production: A Review

Stech

Kubick

2015

Antibodies

View full text Add to dashboard Cite

Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

show abstract

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Cited by 71 publications

References 66 publications

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Intein mediated high throughput screening for bispecific antibodies

Cell-Free Synthesis Meets Antibody Production: A Review

Contact Info

Product

Resources

About