Efficient mAb production in CHO cells with optimized signal peptide, codon, and UTR.

Antibody drugs have been used to treat a number of diseases successfully. Producing antibodies with high yield and quality is necessary for clinical applications of antibodies. For a candidate molecule, optimization of a vector to produce sufficient yield and an accurate primary structure is indispensable in the early stage of the production process development. It is especially important to maintain the fidelity of N-terminal sequence. In order to produce antibodies with a high yield and accurate N-terminal, the expression vector was systematically optimized in this study. First, the heavy chain and light chain were co-expressed in Chinese hamster ovary (CHO) cells with different signal peptides. Mass spectrometry (MS) revealed that signal peptides Esp-K, Bsp-H, and 8Hsp-H were accurately deleted from mature antibodies. Further, the yield was doubled by codon optimization and increased by 50% with the presence of untranslated regions (UTR). The combination of UTR with optimal codon and signal peptide to form an expression vector resulted in yield improvement of 150% and correct N-terminal sequences. Moreover, the main product peak was above 98% as assessed by size-exclusion chromatography (SEC). Additionally, the bioactivity of products made from optimized transient gene expression (TGE) was almost identical to the standard sample. The production efficiency and product quality from the identified TGE optimization strategy was further demonstrated through application to two other antibodies. The expression level of SGE (stable gene expression) can also be improved effectively with this optimization strategy. In conclusion, vector optimization via combination of optimized signal peptide, codon, and UTR is an alternative approach for efficient antibody production with high fidelity N-terminal sequence in CHO cells.

Risedronate-functionalized manganese-hydroxyapatite amorphous particles: A potent adjuvant for subunit vaccines and cancer immunotherapy.

The cGAS-STING pathway and the Mevalonate Pathway are druggable targets for vaccine adjuvant discovery. Manganese (Mn) and bisphosphonates are known to exert adjuvant effects by targeting these two pathways, respectively. This study found the synergistic potential of the two pathways in enhancing immune response. Risedronate (Ris) significantly amplified the Mn adjuvant early antibody response by 166-fold and fortified its cellular immunity. However, direct combination of Mn2+ and Ris resulted in increased adjuvant toxicity (40% mouse mortality). By the combination of doping property of hydroxyapatite (HA) and its high affinity for Ris, we designed Ris-functionalized Mn-HA micro-nanoparticles as an organic-inorganic hybrid adjuvant, named MnHARis. MnHARis alleviated adjuvant toxicity (100% vs. 60% survival rate) and exhibited good long-term stability. When formulated with the varicella-zoster virus glycoprotein E (gE) antigen, MnHARis triggered a 274.3-fold increase in IgG titers and a 61.3-fold surge in neutralization titers while maintaining a better long-term humoral immunity compared to the aluminum adjuvant. Its efficacy spanned other antigens, including ovalbumin, HPV18 VLP, and SARS-CoV-2 spike protein. Notably, the cellular immunity elicited by the group of gE + MnHARis was comparable to the renowned Shingrix®. Moreover, intratumoral co-administration with an anti-trophoblast cell surface antigen 2 nanobody revealed synergistic antitumor capabilities. These findings underscore the potential of MnHARis as a potent adjuvant for augmenting vaccine immune responses and improving cancer immunotherapy outcomes.

HBsAg and TLR7/8 dual-targeting antibody-drug conjugates induce sustained anti-HBV activity in AAV/HBV mice: a preliminary study.

Hepatitis B virus (HBV) infection is a significant global health concern due to elevated immunosuppressive viral antigen levels, the host immune system's inability to manage HBV, and the liver's immunosuppressive conditions. While immunotherapies utilizing broadly reactive HBV neutralizing antibodies present potential due to their antiviral capabilities and Fc-dependent vaccinal effects, they necessitate prolonged and frequent dosing to achieve optimal therapeutic outcomes. Toll-like receptor 7/8 (TLR7/8) agonists have been demonstrated promise for the cure of chronic hepatitis B, but their systemic use often leads to intense side effects. In this study, we introduced immune-stimulating antibody conjugates which consist of TLR7/8 agonists 1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-imidazo[4,5-c]quinolin-4-amine (IMDQ) linked to an anti-hepatitis B surface antigen (HBsAg) antibody 129G1, and designated as 129G1-IMDQ. Our preliminary study highlights that 129G1-IMDQ can prompt robust and sustained anti-HBsAg specific reactions with short-term administration. This underscores the conjugate's potential as an effective strategy for HBsAg clearance and seroconversion, offering a fresh perspective for a practical therapeutic approach in the functional cure of CHB. Highlights: HBV-neutralizing antibody 129G1 was linked with a TLR7/8 agonist small molecule compound IMDQ.Treatment with 129G1-IMDQ has shown significant promise in lowering HBsAg levels in AAV/HBV mice.129G1-IMDQ were eliciting a strong and lasting anti-HBsAg immune response after short-term treatment in AAV/HBV mice.

A glycoengineered therapeutic anti-HBV antibody that allows increased HBsAg immunoclearance improves HBV suppression in vivo.

Introduction: The effective and persistent suppression of hepatitis B surface antigen (HBsAg) in patients with chronic HBV infection (CHB) is considered to be a promising approach to achieve a functional cure of hepatitis B. In our previous study, we found that the antibody E6F6 can clear HBsAg through FcγR-mediated phagocytosis, and its humanized form (huE6F6 antibody) is expected to be a new tool for the treatment of CHB. Previous studies have shown that the glycosylation of Fc segments affects the binding of antibodies to FcγR and thus affects the biological activity of antibodies in vivo. Methods: To further improve the therapeutic potential of huE6F6, in this study, we defucosylated huE6F6 (huE6F6-fuc-), preliminarily explored the developability of this molecule, and studied the therapeutic potential of this molecule and its underlying mechanism in vitro and in vivo models. Results: huE6F6-fuc- has desirable physicochemical properties. Compared with huE6F6-wt, huE6F6-fuc- administration resulted in a stronger viral clearance in vivo. Meanwhile, huE6F6-fuc- keep a similar neutralization activity and binding activity to huE6F6-wt in vitro. Immunological analyses suggested that huE6F6-fuc- exhibited enhanced binding to hCD32b and hCD16b, which mainly contributed to its enhanced therapeutic activity in vivo. Conclusions: In summary, the huE6F6-fuc- molecule that was developed in this study, which has desirable developability, can clear HBsAg more efficiently in vivo, providing a promising treatment for CHB patients. Our study provides new guidance for antibody engineering in other disease fields.

Efficient development of a stable cell pool for antibody production using a single plasmid.

Therapeutic antibodies are the fastest growing group of biopharmaceuticals. Evaluation of drug candidates requires a sufficient amount of antibodies. Production of antibodies with stable cell pools is an efficient strategy to produce grams of proteins for drug candidate selection. Many methods have been described for developing stable cell pools for antibody expression. However, most of the reported methods are laborious due to the low frequency of high producers. In this study, we determined optimal vectors and screening parameters to develop a strategy for efficient construction of stable antibody expressing cell pools. The cell pool constructed using the optimized strategy consistently yielded a higher expression titer, up to 10-fold improvement. Further, this method resulted in a higher ratio of the cell pools with the main product peak above 95% as assessed by size-exclusion chromatography. High producers could be obtained by means of screening five 96-well plates. This strategy will greatly reduce clone-screening size during Clinical Lead Selection. This study provides a platform with efficient design of plasmids and screening strategies for significant cost and labour savings in high expression of two-subunit proteins such as antibodies.

Novel electrochemical biosensor based on functional composite nanofibers for sensitive detection of p53 tumor suppressor gene.

A novel electrochemical biosensor based on functional composite nanofibers for sensitive hybridization detection of p53 tumor suppressor using methylene blue (MB) as an electrochemical indicator is developed. The carboxylated multi-walled carbon nanotubes (MWNTs) doped nylon 6 (PA6) composite nanofibers (MWNTs-PA6) was prepared using electrospinning, which served as the nanosized backbone for pyrrole (Py) electropolymerization. The functional composite nanofibers (MWNTs-PA6-PPy) used as supporting scaffolds for ssDNA immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. The biosensor displayed good sensitivity and specificity. The target wild type p53 sequence (wtp53) can be detected as low as 50 fM and the discrimination is up to 57.5% between the wtp53 and the mutant type p53 sequence (mtp53). It holds promise for the early diagnosis of cancer development and monitoring of patient therapy.