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Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

The cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and subsets of frontotemporal dementia (FTD). In order to reduce TDP-43 pathology, we generated single-chain (scFv) antibodies against the RNA recognition motif 1 (RRM1) of TDP-43, which is involved in abnormal protein self-aggregation and interaction with p65 NF-κB. Virus-mediated delivery into the nervous system of a scFv antibody, named VH7Vk9, reduced microgliosis in a mouse model of acute neuroinflammation and mitigated cognitive impairment, motor defects, TDP-43 proteinopathy, and neuroinflammation in transgenic mice expressing ALS-linked TDP-43 mutations. These results suggest that antibodies targeting the RRM1 domain of TDP-43 might provide new therapeutic avenues for the treatment of ALS and FTD.

Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called “difficult-to-express” proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.

Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

Background Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. Results The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10 10 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. Conclusion The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Pathogenic animal and human viruses present a growing and persistent threat to humans worldwide. Ebola virus (EBOV) causes zoonosis in humans. Here, two structurally different anti-Ebola 13F6 antibodies, recognizing the heavily glycosylated mucin-like domain (MLD) of the glycoprotein (GP), were expressed in transgenic Nicotiana tabacum plants and designed as inexpensive and effective diagnostic antibodies against Ebola virus disease (EVD). The first was anti-EBOV 13F6 full size antibody with heavy chain (HC) and light chain (LC) (monoclonal antibody, mAb 13F6-FULL), while the second was a large single-chain (LSC) antibody (mAb 13F6-LSC). mAb 13F6-LSC was constructed by linking the 13F6 LC variable region (VL) with the HC of mAb 13F6-FULL using a peptide linker and extended to the C-terminus using the endoplasmic reticulum (ER) retention motif KDEL. Agrobacterium-mediated plant transformation was employed to express the antibodies in N. tabacum. PCR, RT-PCR, and immunoblot analyses confirmed the gene insertion, transcription, and protein expression of these antibodies, respectively. The antibodies tagged with the KDEL motif displayed high-mannose type N-glycan structures and efficient binding to EBOV-like particles (VLPs). Thus, various forms of anti-EBOV plant-derived mAbs 13F6-FULL and LSC with efficient binding affinity to EBOV VLP can be produced in the plant system.