A bispecific antibody targeting HER2 and CLDN18.2 eliminates gastric cancer cells expressing dual antigens by enhancing the immune effector function.

Gastric cancer (GC) is widely regarded as one of the toughest cancers to treat. Trastuzumab, which targets the human epidermal growth factor receptor 2 (HER2) for GC treatment, has demonstrated clinical success. However, these patients have a high likelihood of developing resistance. Additionally, Claudin18.2 (CLDN18.2) is a promising emerging target for GC treatment. Therefore, therapies that simultaneously target both HER2 and CLDN18.2 targets are of great significance. Here, we constructed a bispecific antibody targeting both HER2 and CLDN18.2 (HC-2G4S; BsAb), which displayed satisfactory purity, thermostability and enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In a tumor spheroids model of GC, BsAb demonstrated greater therapeutic efficacy than monoclonal antibodies (mAb) or combination treatment strategies. We propose that the enhanced anti-tumor potency of BsAbs in vivo is due to the monovalent binding of single-chain antibodies to more targets due to weaker affinity, resulting in a more potent immune effect function. Therefore, HC-2G4S could be a productive agent for treating GC that is HER2-positive, CLDN18.2-positive, or both, with the potential to overcome trastuzumab resistance and provide significant clinical benefits and expanded indications.

Proton pump inhibitors interfere with the anti-tumor potency of RC48ADC.

Antibody-drug conjugates (ADCs) are a promising modality for cancers, but the interaction between them and proton pump inhibitors (PPIs), the common adjuvant drugs for cancer treatment, has not been understood. Here, the interactions between PPIs and RC48ADC, a novel HER2-targeting ADC, were quantified in vitro. CCK-8 assay showed that RC48ADC displayed a significant inhibitory effect on the proliferation of SK-BR-3, NCI-N87 and SK-OV-3 cells with the IC50 values of 4.91 ± 1.15 ng/mL, 14.54 ± 0.85 ng/mL and 11.28 ± 0.68 ng/mL respectively. PPIs alone had no significant anti-tumor effect in the dose range of 1.37-1000 ng/mL. When used together, PPIs inhibited the anti-tumor activity of RC48ADC in a dose-dependent manner. And 1000 ng/mL (~Cmax) PPIs significantly recovered RC48ADC-inhibited cell proliferation by (32.85 ± 2.81) % (p < 0.05). However, cimetidine, a non-PPIs gastric acid secretion inhibitor, had no significant inhibitory effect on RC48ADC. Furthermore, omeprazole, rather than cimetidine, significantly reduced the activity of vacuolar H+-ATPase and Cathepsin B compared with the control cells. These results, if confirmed in vivo, indicate that PPIs are antagonists of RC48ADC, even all ADCs, appearing to be due to inhibition of vacuolar H+-ATPase activity. Moreover, cimetidine combined with ADCs instead of PPIs can prevent an adverse drug interaction.

Fluctuations in dissolved oxygen concentration during a CHO cell culture process affects monoclonal antibody productivity and the sulfhydryl-drug conjugation process.

During a CHO cell culture production process, important parameters are generally well controlled by a feedback mechanism (PID loop) in order to ensure consistency in both productivity and product quality. These parameters typically include pH, dissolved oxygen (DO), and temperature. While most of these parameters are very well controlled within their specific deadband, stable DO control can be challenging. Oscillations in DO concentration are not uncommon and these fluctuations can be exacerbated with an efficient mass transfer aeration strategy. In this study, where an IgG2 producing cell line was used, we observed increased lactate accumulation accompanied by decreased titer production in lots with fluctuations in DO concentration (DOF ) when compared with lots with stable DO control (DOS ). We demonstrate that DOF had a greater impact on performance with respect to titer production and lactate accumulation than DO setpoint. Furthermore, we report that estimated specific oxygen uptake rates (qOURs) were lower in DOF lots when compared with DOS lots. We also report that purified mAb sourced from DOF lots yielded lower drug-to-antibody ratio (DAR) after the sulfhydryl-targeted maleimide conjugation process when equivalent reducing agent was used. All mAb lots were within the analytical specifications for release, though a slight increase in measureable trisulfides were observed in DOF mAb lots. DO control aimed to minimize fluctuations around DO setpoint was essential for us to produce consistent DAR without adjusting the conjugation process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1427-1437, 2018.

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study.

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.

Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate.

High levels of glycerol significantly inhibit cell growth and 1,3-propanediol (1,3-PD) production in anaerobic glycerol fermentation by genetically engineered Escherichia coli (E. coli) strains expressing genes from the Klebsiella pneumoniae dha (K.pneumoniae) regulon. We have previously demonstrated that 1,3-PD production by the engineered E. coli can be improved by reducing the accumulation of methylglyoxal. This study focuses on investigation of another lesser-known metabolite in the pathways related to 1,3-PD production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in the absence of an exogenous acceptor, the engineered E. coli strains have intracellular G3P levels that are significantly higher than those in K. pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. coli strains, the G3P levels increase with increasing glycerol concentrations, whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. Addition of fumarate, which can stimulate activity of anaerobic G3P dehydrogenase, into the fermentation medium led to a greater than 30-fold increase in the specific activity of anaerobic G3P dehydrogenase and a significant decrease in concentrations of intracellular G3P and resulted in better cell growth and an improved production of 1,3-PD. This indicates that the low activity of G3P dehydrogenase in the absence of an exogenous electron acceptor is one of the reasons for G3P accumulation. In addition, spent media from E.coli Lin61, a glycerol kinase (responsible for conversion of glycerol to G3P) mutant, contains greatly decreased concentrations of G3P and shows improved production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain E. coli K10. This further suggests that G3P accumulation is one of the reasons for the inhibition of 1,3-PD production during anaerobic fermentation.