Full-length recombinant antibodies from Escherichia coli : production, characterization, effector function (Fc) engineering, and clinical evaluation

Abstract

Although several antibody fragments and antibody fragment-fusion proteins produced in Escherichia coli ( E. coli ) are approved as therapeutics for various human diseases, a full-length monoclonal or a bispecific antibody produced in E. coli has not yet been approved. The past decade witnessed substantial progress in expression of full-length antibodies in the E. coli cytoplasm and periplasm, as well as in cell-free expression systems. The equivalency of E. coli -produced aglycosylated antibodies and their mammalian cell-produced counterparts, with respect to biochemical and biophysical properties, including antigen binding, in vitro and in vivo serum stability, pharmacokinetics, and in vivo serum half-life, has been demonstrated. Extensive engineering of the Fc domain of aglycosylated antibodies enables recruitment of various effector functions, despite the lack of N -linked glycans. This review summarizes recent research, preclinical advancements, and clinical development of E. coli -produced aglycosylated therapeutic antibodies as monoclonal, bispecific, and antibody-drug conjugates for use in autoimmune, oncology, and immuno-oncology areas. Abbreviations: ADA Anti-drug antibody; ADCC Antibody-dependent cellular cytotoxicity; ADCP Antibody-dependent cellular phagocytosis; ADC Antibody-drug conjugate; aFc Aglycosylated Fc; AMD Age-related macular degeneration aTTP Acquired thrombotic thrombocytopenic purpura; BCMA B-cell maturation antigen; BLA Biologics license application; BsAb Bispecific antibody; C1q Complement protein C1q; CDC Complement-dependent cytotoxicity; CDCC Complement-dependent cellular cytotoxicity; CDCP Complement-dependent cellular phagocytosis; CEX Cation exchange chromatography; CFPS Cell-free protein expression; CHO Chinese Hamster Ovary; CH1-3 Constant heavy chain 1-3; CL Constant light chain; DLBCL Diffuse large B-cell lymphoma; DAR Drug antibody ratio; DC Dendritic cell; dsFv Disulfide-stabilized Fv; EU European Union; EGFR Epidermal growth factor receptor; E. coli Escherichia coli; EpCAM Epithelial cell adhesion molecule; Fab Fragment antigen binding; FACS Fluorescence activated cell sorting; Fc Fragment crystallizable; FcRn Neonatal Fc receptor; FcɣRs Fc gamma receptors; FDA Food and Drug Administration; FL-IgG Full-length immunoglobulin; Fv Fragment variable; FolRαa Folate receptor alpha; gFc Glycosylated Fc; GM-CSF Granulocyte macrophage-colony stimulating factor; GPx7 Human peroxidase 7; HCL Hairy cell leukemia; HIV Human immunodeficiency virusl; HER2 Human epidermal growth factor receptor 2; HGF Hepatocyte growth factor; HIC Hydrophobic interaction chromatography; HLA Human leukocyte antigen; IBs Inclusion bodies; IgG1-4 Immunoglobulin 1-4; IP Intraperitoneal; ITC Isothermal titration calorimetry; ITP Immune...

Identification of subtype recognition and prognosis model characterized by Antibody-dependent cell phagocytosis related genes in breast cancer

“…The rise of ADCP-related research has revolutionized cancer treatment. Changes, through ADCP-related targets can obviously improve the OS rate of cancer patients (32)(33)(34)(35)(36) .…”

Bioprocessing and the Production of Antiviral Biologics in the Prevention and Treatment of Viral Infectious Disease

“…Interferon Produced Monoclonal Antibody Produced Comments Bacterial E. coli, Bacillus subtillis [33] IFNα-2a, IFNα-2b, IFNα-2c, IFNα-con-1, INFβ-1b, INFγ-1b [92] No approved products Lack of PTMs limits the production of full-length mAbs, though research is ongoing [118] Fungal P. pastoris, S. cerevisiae Filamentous fungi Aspergillus species, Trichoderma reesei and Neurospora crassa [33] IFNα-2b [97] No approved products Glycoengineered yeast can produce interferons and functional full-length mAbs, however extensive clinical research is still required [119] Plant Tobacco (Nicotiana benthamiana) IFN-α2b, IFN-γ [97] Polyclonal antibody of 3 mAB ZMapp (Ebola) [33,120] Very-large-scale processes for plant production are still in development and require substantial investments [ No approved products Offers a green mode of production, affected by low yield.…”

State-of-the-Art Approaches to Heterologous Expression of Bispecific Antibodies Targeting Solid Tumors

“…Relatively simple proteins, such as antibody fragments fused in a single polypeptide chain, can be produced in prokaryotic hosts, usually Escherichia coli cells [42]. More complex proteins, e.g., full-size antibodies consisting of several polypeptide chains, are typically synthesized in eukaryotic, mainly mammalian, cell lines [43].…”

“…Production of bsAbs in E. coli is also possible; however, it can be even more challenging than production of monospecific antibodies due to the complex structure of bsAbs and the need to maintain the natural antibody architecture. According to the recent study [42], several antibody fragments and antibody fragment fusion proteins produced in E. coli have been approved for the treatment of various human diseases, while full-length mAbs and bsAbs expressed in E. coli have not yet been approved.…”