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A More Comprehensive New Approach to Antibody Expression—Cell-Free Protein Expression (Surprise at the End)
Release date:
2024-09-14
Global research on antibody therapeutics is thriving, and China’s antibody drug development is likewise in full swing. At present, recombinant antibody production primarily relies on mammalian cell expression systems such as CHO cells, while Escherichia coli is typically used only for expressing antibody fragments. Although the CHO cell system can produce full-length antibodies, it still faces challenges including difficult cell culture, cumbersome procedures, lengthy expression cycles, and high costs.
Perotin’s proprietary cell-free protein synthesis (CFPS) system supports the autonomous assembly of multiple polypeptide chains, enabling soluble expression in the supernatant of antibody fragments such as VHHs and scFvs, as well as full-length IgG antibodies. The Perotin CFPS system also facilitates the rapid production of bispecific antibodies, significantly shortening the development timeline for bispecific therapeutics. Moreover, requiring no cell culture, the Perotin CFPS system is compatible with high-throughput liquid-handling workstations, enabling large-scale antibody screening.
Recently, Perotin Bio has successfully developed a groundbreaking technology that employs CFPS to achieve site-specific incorporation of non‑natural amino acids (nnAAs) into protein sequences, which is poised to significantly elevate China’s R&D capabilities in next‑generation antibody‑drug conjugates (ADCs).
Bispecific antibody
Bispecific antibodies (bsAbs) are antibody molecules capable of simultaneously recognizing two distinct epitopes on the same or different antigens. bsAbs can redirect effector cells and effector molecules, and their applications are broad, making them potential therapeutic agents for a variety of diseases, including cancer, chronic inflammatory disorders, autoimmune conditions, neurodegenerative diseases, bleeding disorders, and infectious diseases. [1] 。
According to reports, there are currently 712 bispecific antibody drugs in active development. [2] . bsAbs exhibit structural diversity, encompassing small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG-like structure, and large multimeric complexes assembled from distinct antigen-binding domains [1].
Figure 1. Schematic diagram of the mechanism of action of bispecific antibodies. [3]
The construction methods for bispecific antibodies are highly diverse, with the KIHs (knobs-into-holes) technology being widely employed. This approach leverages the differences in the sizes of amino acid side chains between two α‑helices to create “knob” and “hole” structures on the CH3 domains of the two heavy chains, thereby promoting heterodimerization and enhancing the purity of the bispecific antibody.
The internationally renowned cell-free technology company Sutro has successfully synthesized the KIH bispecific antibody using its proprietary cell-free expression system, Xpress CF. [4] The efficient expression of KIH depends on the roughly equal expression of both the knob and hole domains, which is difficult to achieve in conventional cell‑based expression systems. In contrast, the CFPS system allows flexible control over the plasmid ratio of knob‑ and hole‑expressing constructs, thereby enabling the high‑yield synthesis of KIH bispecific antibodies.
Furthermore, the CFPS system enables the co-expression of multiple polypeptide chains, eliminating the cumbersome procedures associated with cellular expression of multiple chains and significantly reducing the time required for bispecific antibody production.
Full-length antibody
The Perotin Bio CFPS system enables the autonomous assembly of multiple peptide chains, allowing not only the expression of antibody Fab fragments but also the production of full-length IgG antibodies. Experimental results demonstrate that the antibodies expressed by the CFPS system exhibit robust immunological activity.
Expression Case
Figure 2. SDS-PAGE analysis of an IgG and its Fab fragment, along with their ELISA activity (CFPS).
VHH, scFv
VHH, or the variable domain of the heavy chain in heavy-chain‑only antibodies—also known as nanobodies—is currently the smallest antigen‑binding unit known. VHH exhibits exceptionally high solubility, resists aggregation, and withstands denaturing conditions such as high temperature, strong acid, and strong alkali, making it widely applicable in the development of therapeutic antibody drugs, diagnostic reagents, affinity purification matrices, and other fields.
scFv, or single-chain variable fragment, is composed of the heavy-chain variable region (VH) and the light-chain variable region (VL), linked by a short peptide of 15–20 amino acids, and does not contain the Fc fragment.
Figure 3 Schematic diagram of the structures of VHH and scFv [5]
Perotin Bio offers antibody molecule expression and purification services based on its proprietary CFPS system, enabling the production of VHHs and scFvs within hours and delivering antibody molecules with 75–90% purity in 1–2 weeks.
Expression Case
Figure 4: SDS-PAGE analysis of two VHHs (left) and an scFv (right) (CFPS)
nnAA定点 insertion and ADC
ADCs consist of a monoclonal antibody, a potent cytotoxic payload, and a linker. Site-specific incorporation of non‑natural amino acids can introduce unique side-chain functional groups, enabling the engineering or modification of antibodies to achieve efficient, site‑specific conjugation of the antibody to the cytotoxic agent. This approach enhances ADC homogeneity and expands the therapeutic window.
Recently, Perotin Bio has successfully developed a groundbreaking technology that employs CFPS to achieve site-specific incorporation of nnAAs into protein sequences, with pAcF insertion efficiencies exceeding 70%. Perotin Bio now offers site-specific incorporation services for p‑acetylphenylalanine (pAcF) and p‑azidophenylalanine (pAzF), along with additional nnAA incorporation assay services, thereby supporting the development of novel ADC therapeutics in China.
For more details, please refer to our previous article: A Domestic First—Site-Specific Incorporation of Non-Natural Amino Acids into Proteins!
A surprise at the end of the text
Anyone who refers a prospective customer to purchase Perotin’s products and services will receive a free RuiXing coffee voucher worth 27 yuan! (Limited to 20 vouchers per day—while supplies last.)
Phone: 0512-67900128
Official website: www.cellfreeprotein.cn
References
[1]BRINKMANN U, KONTERMANN R E. The making of bispecific antibodies[M/OL]//mAbs. Taylor and Francis Inc., 2017: 182-212. DOI:10.1080/19420862.2016.1268307.
[2] Mao Kaiyun, Li Rong, Li Dandan, et al. Analysis of the Global Landscape of Bispecific Antibody Drug Development [J]. Chinese Journal of Biotechnology, 2021, 41(11): 110–118.
[3]SUURS F V., LUB-DE HOOGE M N, DE VRIES E G E, et al. A review of bispecific antibodies and antibody constructs in oncology and clinical challenges[M/OL]//Pharmacology and Therapeutics. Elsevier Inc., 2019: 103-119. DOI:10.1016/j.pharmthera.2019.04.006.
[4]XU Y, LEE J, TRAN C, et al. Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system[J/OL]. mAbs, 2015, 7(1): 231-242. DOI:10.4161/19420862.2015.989013.
[5]BANNAS P, HAMBACH J, KOCH-NOLTE F. Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics[M/OL]//Frontiers in Immunology. Frontiers Media S.A., 2017. DOI:10.3389/fimmu.2017.01603.
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