Production Of Human Antibodies Research Articles

A Brief Chronicle of Antibody Research and Technological Advances.

This review briefly traces the historical development of antibody research and related technologies. The path from early perceptions of immunity to the emergence of modern immunotherapy has been marked by pivotal discoveries and technological advances. Early insights into immunity led to the development of vaccination and serotherapy. The elucidation of antibody structure and function paved the way for monoclonal antibody technology and its application in diagnosis and therapy. Breakthroughs in genetic engineering have enabled the production of humanized antibodies and the advances in Fc engineering, thereby increasing therapeutic efficacy. The discovery of immune checkpoints and cytokines revolutionized the treatment of cancer and autoimmune diseases. The field continues to evolve rapidly with the advent of antibody-drug conjugates, bispecific antibodies, and CAR T-cell therapies. As we face global health challenges, antibody research remains at the forefront of medical innovation and offers promising solutions for the future.

The aryl hydrocarbon receptor differentially modulates the expression profile of antibody isotypes in a human B-cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cμ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cμ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.

Production of Anti-Recombinant Human Insulin Antibody and Validation by Indirect ELISA

Human insulin potential has become an interest and is important in maintaining the success of therapyin patients with the availability of chemical-based analytical methods, however, only a few have beenusing immunoassays. This study aimed to produce IgG polyclonal antibodies from rabbits immunizedwith 1 mg/mL rhINS subcutaneously and validated by indirect ELISA. Antibody was precipitated andfractioned on a HiTrap® Protein A HP column before being quantified with a UV spectrophotometerat λ 280 nm. The characterization was conducted by Dot Blot test on a BCIP-NBT substrate, as well asSDS-PAGE and Western Blot with polyacrylamide gel concentrations of 7.5% and 17.5%. Validationwas performed using solutions containing glycerol and m-cresol as matrices spiked with rhINS. Thelinearity test in the rhINS concentration range of 80.11-200.28 μg/mL (r = 0.99) showed the linearresult. The accuracy and precision obtained an average of 99.11%±5.01 and 3.91%, while the LODand LOQ were 22.05 μg/mL and 73.51 μg/mL, respectively. Human insulin was stable at 2-8oC for 24hours (α: 0.05, ANOVA). In conclusion, in-house produced IgG polyclonal antibodies and goat anti-IgG peroxidase conjugate can be used for routine testing of human insulin.

The Effect of Photoperiod Duration on Humoral Innate and Humoral Adaptive Immune Responsiveness in Campbell’s Dwarf Hamster

We studied the humoral innate immune responsiveness (HII), i.e. a hemolysis of rabbit erythrocytes by complement proteins, and adaptive humoral (antibody production) immune responsiveness (AHI) to SRBC, morphological and hormonal reproductive characteristics and stress level in male Campbell hamsters kept under long-day (LD; 16D: 8N) and short-day (SD; 8D: 16N) photoperiods. The results indicate lower HII, but not AHI in SD. In SD males we observed body mass, anogenital distance, midventral gland size and level of testosterone in the peripheral blood (but not the level of cortisol) decreased. Comparison of SD non-responders, SD responders and LD individuals demonstrated a statistically significant increase of HII in SD photosensitive hamsters, but not in non-responders compared to LD. There was no link between HII and AHI, which indicates an independent photoperiodic responsiveness of different branches of the immune system in Campbell’s hamster.

Phage antibody library technology in tumor therapy: a review

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.

Characterization of the novel glucose-methanol-choline (GMC) oxidoreductase EnOXIO1 in Eimeria necatrix

Eimeria species are intracellular obligate parasites, among the most common pathogens affecting the intensive poultry industry. Oxidoreductases are members of a class of proteins with redox activity and are widely found in apicomplexan protozoans. However, there have been few reports related to Eimeria species. In this study, total RNA was extracted from the gametocytes of E. necatrix Yangzhou strain to amplify the EnOXIO1 gene using reverse-transcription polymerase chain reaction. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into Escherichia coli BL21(DE3), and the recombinant protein rEnOXIO1 was expressed by induction with isopropyl ß-D-1-thiogalactopyranoside. The full length EnOXIO1 gene was 2535 bp encoding 844 amino acids, and the EnOXIO1 protein had a molecular weight of about 100 kDa and was mainly expressed in inclusion bodies. Western blot analysis indicated that the rEnOXIO1 protein had good antigenicity and cross-reactivity and was specifically recognized by a 6 ×HIS labeled monoclonal antibody, mouse anti-recombinant protein polyclonal antibody, and recovery serum from chickens infected with E. necatrix, E. acervulina, and E. tenella sporulated oocysts. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein was mainly located on the wall-forming bodies in gametocytes and played an important role in the formation of the oocyst wall. Quantitative PCR analysis revealed that transcript levels of EnOXIO1 were highest in the gametocyte stage. Protein expression levels of EnOXIO1 were higher in the gametocyte stage than in other developmental stages according to western blot analysis. Vaccination of chickens against E. necatrix was achieved with recombinant protein rEnOXIO1, which triggered humoral immunity and antibody production, increased average body weight gain, reduced oocyst output and alleviated lesions after E. necatrix infection. The highest ACI value (172.36) was observed in chickens that received 200 μg rEnOXIO1 compared with other immunization groups.

Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats.

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 μg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.

Chimeric Hepatitis B core virus-like particles harboring SARS-CoV2 epitope elicit a humoral immune response in mice

BackgroundVirus-like particles are an interesting vector platform for vaccine development. Particularly, Hepatitis B virus core antigen has been used as a promising VLP platform. It is highly expressed in different recombinant expression systems, such as E. coli, and self-assembled in vitro. It effectively improves the immunogenicity of foreign antigenic epitopes on its surface. Various foreign antigens from bacteria, viruses, and protozoa can be genetically inserted into such nanoparticles. The effective immunogenicity due to VLP vaccines has been reported. However, no research has been performed on the SARS-CoV2 vaccine within this unique platform through genetic engineering. Considering the high yield of target proteins, low cost of production, and feasibility of scaling up, E. coli is an outstanding expression platform to develop such vaccines. Therefore, in this investigation, we planned to study and develop a unique HBc VLP-based vaccine against SARS-Cov2 utilizing the E. coli expression system due to its importance.ResultsInsertion of the selected epitope was done into the major immunodominant region (MIR) of truncated (149 residues) hepatitis B core capsid protein. The chimeric protein was constructed in PET28a+ and expressed through the bacterial E. coli BL21 expression system. However, the protein was expressed in inclusion body forms and extracted following urea denaturation from the insoluble phase. Following the extraction, the vaccine protein was purified using Ni2 + iminodiacetic acid (IDA) affinity chromatography. SDS-PAGE and western blotting were used to confirm the protein expression. Regarding the denaturation step, the unavoidable refolding process was carried out, so that the chimeric VLP reassembled in native conformation. Based on the transmission electron microscopy (TEM) analysis, the HBC VLP was successfully assembled. Confirming the assembled chimeric VLP, we explored the immunogenic effectivity of the vaccine through mice immunization with two-dose vaccination with and without adjuvant. The utilization of adjuvant was suggested to assess the effect of adjuvant on improving the immune elicitation of chimeric VLP-based vaccine. Immunization analysis based on anti-spike specific IgG antibody showed a significant increase in antibody production in harvested serum from immunized mice with HBc-VLP harboring antigenic epitope compared to HBc-VLP- and PBS-injected mice.ConclusionsThe results approved the successful production and the effectiveness of the vaccine in terms of humoral IgG antibody production. Therefore, this platform can be considered a promising strategy for developing safe and reasonable vaccines; however, more complementary immunological evaluations are needed.

Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

BackgroundThe biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production. ResultsWe have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated.The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs.Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

Evolutive emergence and divergence of an Ig regulatory node: An environmental sensor getting cues from the aryl hydrocarbon receptor?

One gene, the immunoglobulin heavy chain (IgH) gene, is responsible for the expression of all the different antibody isotypes. Transcriptional regulation of the IgH gene is complex and involves several regulatory elements including a large element at the 3' end of the IgH gene locus (3'RR). Animal models have demonstrated an essential role of the 3'RR in the ability of B cells to express high affinity antibodies and to express different antibody classes. Additionally, environmental chemicals such as aryl hydrocarbon receptor (AhR) ligands modulate mouse 3'RR activity that mirrors the effects of these chemicals on antibody production and immunocompetence in mouse models. Although first discovered as a mediator of the toxicity induced by the high affinity ligand 2,3,7,8-tetracholordibenzo-p-dioxin (dioxin), understanding of the AhR has expanded to a physiological role in preserving homeostasis and maintaining immunocompetence. We posit that the AhR also plays a role in human antibody production and that the 3'RR is not only an IgH regulatory node but also an environmental sensor receiving signals through intrinsic and extrinsic pathways, including the AhR. This review will 1) highlight the emerging role of the AhR as a key transducer between environmental signals and altered immune function; 2) examine the current state of knowledge regarding IgH gene regulation and the role of the AhR in modulation of Ig production; 3) describe the evolution of the IgH gene that resulted in species and population differences; and 4) explore the evidence supporting the environmental sensing capacity of the 3'RR and the AhR as a transducer of these cues. This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3'RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions, including emerging infections such as SARS-CoV-2.

Технологии создания вируснейтрализующих антител человека на примере SARS-CoV-2

Monoclonal antibodies (mAbs) are the most promising and most intensively replenished type of bioactive pharmaceuticals. Currently, there are over 100 different mAbs approved by the FDA and other regulating agencies for treatment of oncological, infectious, systemic, autoimmune and other diseases. Design of antibodies neutralizing pathogens of socially significant infections, such as HIV, hepatitis viruses, SARS-CoV-2, is a separate direction. The SARS-CoV-2 pandemic has shown how urgent it is to have a technological platform enabling production of fully human antibodies. The development of recombinant DNA technology and antibody phage display enabled compilation of libraries of antigen-binding fragments and screening with target antigens. This review discusses the advantages and disadvantages of phage display, including use of single-domain antibody technology based on the heavy chain variable domain. We describe the state-of-the-art (and practical results of its application) technology enabling production of human antibodies by sorting and sequencing the genome of individual memory B cells, using monoclonal virus-neutralizing antibodies against SARS-CoV-2 as an example. The prospects of further development of the recombinant human antibody production technology are discussed; in particular, we consider creation of sequences of variable fragments of antibodies with the help of artificial intelligence.

A novel platform for the production of autologous human antibodies.

At Research Genetic Cancer Centre, we have developed a novel method for the production of human monoclonal antibodies against a specific antigen of our choice (c-met) using isolated human blood cells. By mimicking nature, dendritic, CD4 and CD19 cells from healthy volunteers were driven towards Th2 immunity. Cell activation was succeeded by a cytokine cocktail, and IgG production was promoted by IgG class switching factors. IgG secretion was determined using both enzyme linked immunosorbent assay (ELISA) and Western blot as well as immunoglobulin heavy chain gamma polypeptide gene expression. Secreted antibody was further purified by affinity column chromatography against c-met peptide. Anti-c-met activity was determined using the purified antibody as primary antibody for c-met detection by ELISA, Western blot and flow cytometry. Finally, anti-c-met antibody efficiency was determined by MCF-7 viability assay. Plasma cell formation and IgG secretion took place after 6 days of culture. Plasma cells produced anti-c-met IgG antibody that significantly decreased MCF-7 breast cancer cell proliferation. To our knowledge, this is the first platform of its kind, generating fully human antibodies-on-demand using patient's own cells, bringing personalized, targeted therapy for cancer one step closer.

Construction of stable mouse artificial chromosome from native mouse chromosome 10 for generation of transchromosomic mice

Mammalian artificial chromosomes derived from native chromosomes have been applied to biomedical research and development by generating cell sources and transchromosomic (Tc) animals. Human artificial chromosome (HAC) is a precedent chromosomal vector which achieved generation of valuable humanized animal models for fully human antibody production and human pharmacokinetics. While humanized Tc animals created by HAC vector have attained significant contributions, there was a potential issue to be addressed regarding stability in mouse tissues, especially highly proliferating hematopoietic cells. Mouse artificial chromosome (MAC) vectors derived from native mouse chromosome 11 demonstrated improved stability, and they were utilized for humanized Tc mouse production as a standard vector. In mouse, however, stability of MAC vector derived from native mouse chromosome other than mouse chromosome 11 remains to be evaluated. To clarify the potential of mouse centromeres in the additional chromosomes, we constructed a new MAC vector from native mouse chromosome 10 to evaluate the stability in Tc mice. The new MAC vector was transmitted through germline and stably maintained in the mouse tissues without any apparent abnormalities. Through this study, the potential of additional mouse centromere was demonstrated for Tc mouse production, and new MAC is expected to be used for various applications.

Protective effect of myricetin, apigenin, and hesperidin pretreatments on cyclophosphamide-induced immunosuppression

Aim: Major side effects of cyclophosphamide administration are immunosuppression and myelosuppression. The immunomodulatory effects of plant bioactive compounds on chemotherapy drug-induced immunosuppression may have significant effects in cancer treatment. For this reason, we investigated the immunomodulatory effect of myricetin, apigenin, and hesperidin in cyclophosphamide-induced immunosuppression in rats. Methods: In our study, a total of 64 rats were used, and divided into eight equal groups. These groups were: control, cyclophosphamide, cyclophosphamide + myricetin (100 mg/kg), cyclophosphamide + myricetin (200 mg/kg), cyclophosphamide + apigenin (100 mg/kg), cyclophosphamide + apigenin (200 mg/kg), cyclophosphamide + hesperidin (100 mg/kg), and cyclophosphamide + hesperidin (200 mg/kg). Myricetin, apigenin, and hesperidin pretreatments were performed for 14 d, while cyclophosphamide application (200 mg/kg) was performed only on the 4th day of the study. Levels of humoral antibody production, quantitative hemolysis, macrophage phagocytosis, splenic lymphocyte proliferation, and natural killer cell cytotoxicity were determined. In addition, we measured pro-inflammatory cytokines, and followed lipid peroxidation and antioxidant markers and examined the histology of bone marrow, liver and spleen in all groups. Results: During cyclophosphamide treatment, all three phytochemicals increased the levels of humoral antibody production, quantitative hemolysis, macrophage phagocytosis, splenic lymphocyte proliferation, antioxidant markers, and natural killer cell cytotoxicity. Moreover, the agents decreased the levels of pro-inflammatory cytokines and mediators, reduced lipid peroxidation markers, and reduced tissue damage in liver, spleen, and bone marrow. Conclusion: Our study demonstrated that myricetin, apigenin, and hesperidin can reduce the immunosuppressive effect of cyclophosphamide by enhancing both innate and adaptive immune responses, and these compounds may be useful immunomodulatory agents during cancer chemotherapy.

Evidence-Based Immunotherapeutic Effects of Herbal Compounds on Humoral Immunity: Ancient and New Approaches.

The human immune system represents a dynamic multiscale system with high complexity in biology. Humoral immunity, as the main branch of adaptive immunity, is characterized by differentiated stages of the B lymphocytes, producing the final product of antibodies that has a diversity of the tuning mechanisms within genetic and epigenetic levels in confrontation with environmental exposures. Disorders because of disturbed humoral immunity are linked with dysregulation of feedback-regulated signaling and the dynamic of immune components that determine the overall response. Food products, mainly herbal components have a significant role in tailoring the immune system micro-ecosystem which can diversify the adaptive nature of humoral immunity. Herein, we review the current evidence-based approaches for the impact of medicinal herbs on humoral immunity signaling and antibody production with a focus on immunotherapeutic applications.

Human Antibody Production in Plants and Challenges

In nature, plants do not produce antibodies intrinsically against the invading foreign molecules. Recently, model plants genetically engineered for the production of antibodies, known as plantibodies. The potential functions of these plantibodies were found to be as effective as the antibodies produced by the immune system of human beings. Since the last decade, many plantibodies were produced and used for various applications in the areas of diagnostic, therapeutic and veterinary sciences. By taking advantage of secretory and endomembrane systems, plants have been employed for large scale production of viable and purified plant proteins. Moreover, the biological products occupy the major portion of pharmaceutical compounds, the technology for the production of plantibodies provides an excellent platform for pharmaceutical industries against pandemics and several other infections and diseases.

Monoclonal Antibodies Treatment

EAntibodies are naturally produced by the immune system when it detects something harmful. Antibodies are the proteins which act to certain infection by attaching themselves to antigens. The synthetic antibodies are produced in the laboratory which act against proteins that attack normal tissues. Production of synthetic antibodies involves introducing human genes that produce antibodies into mice or any another suitable mammal and later those mice are vaccinated with the antigen for which antibodies are required to be produced, which leads to the production of desired human antibody by the immune cells of the mice. These synthetic antibodies which are produced in the lab are termed as monoclonal antibodies which are synthesized from cloned immune cells. The use of monoclonal antibodies in treating a disease is called immunotherapy where each type of monoclonal antibody is produced to target a specific antigen in the body

Lipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines

The use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine. Using a plasmid encoding an anti-poxvirus monoclonal antibody (as a reporter of protein expression), we showed that improved immunogenicity is likely due to increased in vivo DNA delivery, resulting in more target protein. Specifically, after four days, up to 30 ng/mL of functional monoclonal antibody were detected in the serum of rabbits injected with the LNP-formulated DNA. We pragmatically applied the technology to the production of human neutralizing antibodies in a transchromosomic (Tc) bovine for use as a passive immunoprophylactic. Production of neutralizing antibody was increased by >10-fold while utilizing 10 times less DNA in the Tc bovine. This work provides a proof-of-concept that LNP formulation of DNA vaccines can be used to produce more potent active vaccines, passive countermeasures (e.g., Tc bovine), and as a means to produce more potent DNA-launched immunotherapies.

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

BackgroundMonoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world’s best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.ResultsAdalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions.ConclusionThese results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient’s welfare.