Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody–drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody–drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.

Antibody–drug conjugates (ADCs) have greatly improved the outcomes of advanced breast tumors. However, the treatment of breast tumors with existing ADCs is still hindered by many issues, such as tumor antigen heterogeneity and drug resistance. Therefore, ADCs against new targets would provide options for the treatment of these challenges. Sortilin-1 (SORT1) may be a promising target for ADC as it is upregulated in breast cancer. To evaluate the possibility of SORT1 as an ADC target, a humanized antibody_8D302 with high affinity against SORT1 was generated. Additionally, 8D302 was conjugated with MMAE and DXd to generate two ADCs_8D302-MMAE and 8D302-DXd, respectively. Both 8D302-MMAE and 8D302-DXd showed effective cytotoxicity against SORT1 positive breast tumor cell lines and induced bystander killing. Consequently, 8D302-MMAE showed relatively better anti-tumor activity than 8D302-DXd both in vitro and in vivo, but 8D302-DXd had superior safety profile and pharmacokinetics profile over 8D302-MMAE. Furthermore, SORT1 induced faster internalization and lysosomal trafficking of antibodies and had a higher turnover compared with HER2. Also, 8D302-DXd exhibited superior cell cytotoxicity and tumor suppression over trastuzumab-DXd, a HER2-targeted ADC. We hypothesize that the high turnover of SORT1 enables SORT1-targeted ADC to be a powerful agent for the treatment of SORT1-positive breast tumor.

Nivolumab, a fully human IgG4 anti-programed cell death 1（PD-1）antibody, is recently one of the most popular and successful therapeutic monoclonal antibodies in clinical use. With the increasing demands for Nivolumab and other therapeutic monoclonal antibodies, the mammary gland bioreactor has been regarded as another choice for the production of recombinant monoclonal antibodies besides mammalian cell culture. Here, we expressed a recombinant human anti-PD-1 antibody in the mammary glands of transgenic mice. Two expression vectors were constructed bearing the heavy and light chains of anti-PD-1 antibody respectively under the control of bovine α_s1-casein promoter. Transgenic mice were then generated by co-microinjection of the two expression cassettes. Three F0 founders with both heavy chain and light chain positive were obtained. Transgenes of both chains were detected to be stably transmitted to the offspring. The recombinant antibody was detected in the milk of transgenic mice with the highest expression level up to 80.52 ± 0.82 mg/L and could specifically binds to the human PD-1 antigen. Therefore, our results suggest the feasibility of anti-PD-1 antibody production in the milk of transgenic animals.

Site-specific integration is an important approach used to address the problem of unstable cell lines in industry. In this study, we observed a reduction in the gene copy number and antibody production in a CHOK1 cell line BA03 capable of high antibody expression. We identified a new integration site named locus 7 in the intron region of the parva gene through sequencing, FISH, and genome walking. We demonstrate that the integration of the exogenous gene at this locus does not affect the transcription of the parva and, therefore, has a minimal impact on cell growth. We designed sgRNA and donor vectors to integrate the etanercept-coding gene into locus 7 and obtained a cell line, SSI-4. We performed a passaged stability study on SSI-4 and proved the possibility of the stable, site-specific integration of exogenous genes at this locus in terms of integration site, copy number, expression level, and cell growth. In summary, our study has identified a new integration site suitable for site-specific integration, which lays the foundation for the subsequent development of site-specific integration cell lines.

Squalene is a natural triterpenoid that is important in medicine, and daily-use chemical and nutraceutical industries. The demand for squalene remains high and constant. Shark liver oil is the source of the compound; however, the source is unsustainable due to the protection of the gradually extinct animal. This study aimed to construct an engineered Escherichia coli strain to produce squalene in a green and sustainable manner. In this study, the activity of several squalene synthases from different species was screened. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used for chemical structure identification. The mevalonate pathway (MVA) was constructed into BL21(DE3) to enhance the supply of farnesyl diphosphate to obtain squalene at a concentration of 69.3 mg/L. IspH was overexpressed in BL21(DE3) to decrease squalene production due to the accumulation of dimethylallyl diphosphate. By balancing the overexpression of ispH and ispG, the production of squalene increased to 298.3 mg/L. The genes of the MVA pathway were further separated upstream and downstream, and constructed into two plasmids with different origins to balance the metabolic flux. By optimizing the expression strength of genes in MVA and 2-C-methyl-d-erythritol-4-phosphate pathway, an engineered strain was finally obtained with squalene production of 974.3 mg/L. By replacing chassis cell BL21(DE3) with XL1-Blue, squalene production was further increased to 1,274 mg/L. Received: 13 April 2023 Accepted: 30 September 2023 Article published online: 01 December 2023 © 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany