Generation of a Novel SORT1×HER2 Bispecific Antibody-Drug Conjugate Targeting HER2-Low-Expression Tumor.

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.

Generation and Characterization of SORT1-Targeted Antibody-Drug Conjugate for the Treatment of SORT1-Positive Breast Tumor.

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.

Medium optimization for high yield production of human serum albumin in Pichia pastoris and its efficient purification.

OBJECTIVE: To improve the yield of recombinant human serum albumin (HSA) in Pichia pastoris by medium optimization and establish the related purification scheme. RESULTS: A simplified version of the generally used buffered glycerol complex medium (BMGY), which contained yeast extract, glycerol and potassium salts, was found to be applicable. By decreasing the salt concentration of basal salt medium (BSM) to half of the original formula further, we achieved a high yield of 17.47 g/L HSA in the supernatant within a 192 h induction, which is the highest rHSA yield ever reported as far as we know. Accompanied with a three-step purification procedure which recovered two thirds of the desired protein at high purity, our work lays a solid foundation for large-scale industrial production of HSA. CONCLUSION: Medium optimization plays a significant role in improving the yield of desired protein, lowering the production cost and helping to explore the producing strain's character.

Expression of a recombinant anti-programed cell death 1 antibody in the mammary gland of transgenic mice.

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 αs1-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.

Expression, purification, and characterization of the Degludec precursor DesB30.

Diabetes is a chronic metabolic disease, for which recombinant human insulin is the most effective and mainstream treatment. DesB30 is an insulin analogue in which the B chain lacks amino acid 30 (Thr) compared with human insulin. This analogue can be used to produce the long-acting insulin Degludec or Detemir. In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The insulin precursor, which is single-stranded, was purified by weak cation exchange chromatograph and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), from which 73.39% of product was recovered at over 95% purity. The double-stranded protein (DesB30) was obtained by digesting the insulin precursor with trypsin. Using preparative RP-HPLC, the product was obtained with over 95% purity. Finally, the structure of DesB30 was confirmed.

High level expression and purification of recombinant human serum albumin in Pichia pastoris.

Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris. A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418. After optimizing the induction conditions and additional supplements, the highest yield of HSA reached 1.6 g/L in a shake flask. Performing high density fermentation further improved the highest yield to 8.86 g/L in a fermenter after 96 h of methanol induction. This result is more promising than the previous reports of industrial applications, which reported the highest yield as 92.29 mg/L/h, considering that the space-time yield of rHSA was doubled. In addition, the desired protein was purified by filtration and Cibacron Blue affinity chromatography, which yielded a 58% recovery of a product that had over a 96% purity. This study reveals that Pichia pastoris is an excellent system for recombinant human serum albumin expression due to its outstanding expression capacity. In addition, the high efficiency level of rHSA production lays a solid foundation for its use in industrial production.

Antibiotics, gut microbiota, environment in early life and type 1 diabetes.

The gut microbiota interact with innate immune cells and play an important role in shaping the immune system. Many factors may influence the composition of the microbiota such as mode of birth, diet, infections and medication including antibiotics. In diseases with a multifactorial etiology, like type 1 diabetes, manipulation and alterations of the microbiota in animal models have been shown to influence the incidence and onset of disease. The microbiota are an important part of the internal environment and understanding how these bacteria interact with the innate immune cells to generate immune tolerance may open up opportunities for development of new therapeutic strategies. In this review, we discuss recent findings in relation to the microbiota, particularly in the context of type 1 diabetes.

Maternal Antibiotic Treatment Protects Offspring from Diabetes Development in Nonobese Diabetic Mice by Generation of Tolerogenic APCs.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that involves the slow, progressive destruction of islet ß cells and loss of insulin production, as a result of interaction with environmental factors, in genetically susceptible individuals. The gut microbiome is established very early in life. Commensal microbiota establish mutualism with the host and form an important part of the environment to which individuals are exposed in the gut, providing nutrients and shaping immune responses. In this study, we studied the impact of targeting most Gram-negative bacteria in the gut of NOD mice at different time points in their life, using a combination of three antibiotics--neomycin, polymyxin B, and streptomycin--on diabetes development. We found that the prenatal period is a critical time for shaping the immune tolerance in the progeny, influencing development of autoimmune diabetes. Prenatal neomycin, polymyxin B, and streptomycin treatment protected NOD mice from diabetes development through alterations in the gut microbiota, as well as induction of tolerogenic APCs, which led to reduced activation of diabetogenic CD8 T cells. Most importantly, we found that the protective effect was age dependent, and the most profound protection was found when the mice were treated before birth. This indicates the importance of the prenatal environment and early exposure to commensal bacteria in shaping the host immune system and health.

Expression, Purification, and Activity of ActhiS, a Thiazole Biosynthesis Enzyme from Acremonium chrysogenum.

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (ADT) using HPLC and 1H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD+ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.

Different immunological responses to early-life antibiotic exposure affecting autoimmune diabetes development in NOD mice.

Environmental factors clearly influence the pathogenesis of Type 1 diabetes, an autoimmune disease. We have studied gut microbiota as important environmental agents that could affect the initiation or progression of type 1 diabetes especially in the prenatal period. We used neomycin, targeting mainly Gram negative or vancomycin, targeting mainly Gram positive bacteria, to treat pregnant NOD mothers and to study autoimmune diabetes development in their offspring. Neomycin-treated offspring were protected from diabetes, while vancomycin-treated offspring had accelerated diabetes development, and both antibiotics caused distinctly different shifts in gut microbiota composition compared with the offspring from untreated control mice. Our study demonstrated that neomycin treatment of pregnant mothers leads to generation of immune-tolerogenic antigen-presenting cells (APCs) in the offspring and these APCs had reduced specific autoantigen-presenting function both in vitro and in vivo. Moreover, the protection from diabetes mediated by tolerogenic APCs was vertically transmissible to the second generation. In contrast, more diabetogenic inflammatory T cells were found in the lymphoid organs of the offspring from the vancomycin-treated pregnant mothers. This change however was not transmitted to the second generation. Our results suggested that prenatal exposure to antibiotic influenced gut bacterial composition at the earliest time point in life and is critical for consequent education of the immune system. As different bacteria can induce different immune responses, understanding these differences and how to generate self-tolerogenic APCs could be important for developing new therapy for type 1 diabetes.

Comparative expression profiling of genes involved in primary metabolism in high-yield and wild-type strains of Acremonium chrysogenum.

Cephalosporin C (CPC) productivity of Acremonium chrysogenum has been improved significantly through classical strain improvement programs. Here, we used transcription and metabolite profiling to address mechanisms underlying CPC production in a high yield (HY) strain. Transcription and metabolite profiling indicated that enzymes involved in amino acid production are higher in abundance in the HY strain. Moreover, results indicate a higher flow of precursors from the glycolysis and gluconeogenesis pathways to serine synthesis at the late stage of fermentation in the HY strain. In addition, less pyruvate would enter the TCA cycle thus favoring valine synthesis. Amino acid production would also benefit from a more active pentose phosphate pathway and γ-amino butyric acid shunt both generating NADPH. Moreover the glyoxylate pathway seems to be more active in the HY strain. These results may provide new leads for CPC strain improvement in industry.

Acthi, a thiazole biosynthesis enzyme, is essential for thiamine biosynthesis and CPC production in Acremonium chrysogenum.

BACKGROUND: The filamentous fungus Acremonium chrysogenum is an important industrial fungus and is used in the production of the ß-lactam antibiotic cephalosporin C. Little is known regarding the molecular and biological mechanisms of how this industrial strain was improved by mutagenesis and molecular breeding. Comparative proteomics is one of the most powerful methods to evaluate the influence of gene expression on metabolite production. RESULTS: In this study, we used comparative proteomics to investigate the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer (HY) strain and a wide-type (WT) strain. We found that the expression levels of thiamine biosynthesis-related enzymes, including the thiazole biosynthesis enzyme (Acthi), pyruvate oxidase, flavin adenine dinucleotide (FAD)-dependent oxidoreductase and sulfur carrier protein-thiS, were up-regulated in the HY strain. An Acthi-silencing mutant of the WT strain grew poorly on chemically defined medium (MMC) in the absence of thiamine, and its growth was recovered on MMC medium supplemented with thiamine. The intracellular thiamine content was changed in the Acthi silencing or over-expression mutants. In addition, we demonstrated that the manipulation of the Acthi gene can affect the hyphal growth of Acremonium chrysogenum, the transcription levels of cephalosporin C biosynthetic genes, the quantification levels of precursor amino acids for cephalosporin C synthesis and the expression levels of thiamine diphosphate-dependent enzymes. Over-expression of Acthi can significantly increase the cephalosporin C yield in both the WT strain and the HY mutant strain. CONCLUSIONS: Using comparative proteomics, four differently expressed proteins were exploited, whose functions may be involved in thiamine diphosphate metabolism. Among these proteins, the thiazole biosynthesis enzyme (ActhiS) may play an important role in cephalosporin C biosynthesis. Our studies suggested that Acthi might be involved in the transcriptional regulation of cephalosporin C biosynthesis. Therefore, the thiamine metabolic pathway could be a potential target for the molecular breeding of this cephalosporin C producer for industrial applications.

Bioconversion of deoxysugar moieties to the biosynthetic intermediates of daunorubicin in an engineered strain of Streptomyces coeruleobidus.

Daunorubicin (DNR) is a representative anthracycline with anti-tumor bioactivity. Its convergent biosynthetic pathway has promoted the research on pursuing novel anthracyclines by combinatorial biosynthesis. SnoaL is a special polyketide cyclase that catalyzes the closure of nogalonic acid methyl ester with the C9-S stereochemistry. In this study, the gene cluster of DNR was cloned, and snoaL was integrated into the DNR biosynthetic pathway for the substitution of dnrD in Streptomyces coeruleobidus DM, which resulted in the production of epi-aklaviketone. The biosynthetic pathway of NDP-4-deacetyl-L-chromose B was then expressed in the engineered strain, which led to the production of corresponding glycosylated anthracycline compounds. Finally, the bioactivities of these engineering strains were evaluated.

TLR5-deficiency controls dendritic cell subset development in an autoimmune diabetes-susceptible model.

Introduction: The incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D. Methods: To fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study. Results: We found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice. Discussion: In summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.

Tlr9 deficiency in B cells leads to obesity by promoting inflammation and gut dysbiosis.

Toll-like receptor 9 (TLR9) recognizes bacterial, viral and self DNA and play an important role in immunity and inflammation. However, the role of TLR9 in obesity is less well-studied. Here, we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-, KO) B6 mice and model obesity using a high-fat diet. Compared with control mice, B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation, weight gain, and impaired glucose and insulin tolerance. Furthermore, the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced, and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition, gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing, we identify altered gut microbial communities including reduced Lachnospiraceae, which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9, Irf4 and Il-10 interconnecting metabolic homeostasis, with the function of B and T cells, and gut microbiota in obesity.

[Effect of expressing of anti-PD-1 antibody in mouse mammary gland on spleen T cells in transgenic mice].

This study aims to investigate the effect of anti-PD-1 antibody expressed in mouse mammary gland on the surface antigen protein of spleen T cells, cytokine expression, spleen CD4+ T cell proliferation and proliferation related pathways of transgenic mice at the cellular level. Transgenic mice expressing anti-human PD-1 antibody at 8 weeks of age without pregnancy and 18 weeks of age with lactation were divided into two groups, with transgenic negative mice in each group as the control. Spleen lymphocytes were extracted and the changes of spleen lymphocytes were detected. Compared with transgenic negative mice, the proportion of effector T cells of spleen T cells in the immune system of transgenic mice with anti-PD-1 antibody expressed in breast increased, the proportion of Treg cells decreased, and the IFN-γ, IL-17 and IL-2 expressed in CD4+ T cells increased in varying degrees. Moreover, IL-4, IL-10 and TGF-ß in CD4+ T cells did not change, nor did some cell surface protein molecules related to T cell stimulate. There was no significant difference in T cell proliferation between transgenic positive and transgenic negative mice. In transgenic positive mice, the expression of phosphorylated proteins in PI3K/Akt/mTOR and RAS/MEK/ERK pathways were partially up-regulated, but the whole pathway was not completely up-regulated. Therefore, it is feasible to use transgenic mice as host to express monoclonal antibodies related to immune system such as anti-PD-1 antibody.

Identification of a New Integration Site and Study on Site-Specific Integration in CHO-K1 Cells.

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.

NLRP6 deficiency expands a novel CD103+ B cell population that confers immune tolerance in NOD mice.

Introduction: Gut microbiota have been linked to modulating susceptibility to Type 1 diabetes; however, there are many ways in which the microbiota interact with host cells, including through microbial ligand binding to intracellular inflammasomes (large multi-subunit proteins) to initiate immune responses. NLRP6, a microbe-recognizing inflammasome protein, is highly expressed by intestinal epithelial cells and can alter susceptibility to cancer, obesity and Crohn's disease; however, the role of NLRP6 in modulating susceptibility to autoimmune diabetes, was previously unknown. Methods: We generated NLRP6-deficient Non-obese diabetic (NOD) mice to study the effect of NLRP6-deficiency on the immune cells and susceptibility to Type 1 diabetes development. Results: NLRP6-deficient mice exhibited an expansion of CD103+ B cells and were protected from type 1 diabetes. Moreover, NLRP6-deficient CD103+ B cells express regulatory markers, secreted higher concentrations of IL-10 and TGFb1 cytokines and suppressed diabetogenic T cell proliferation, compared to NLRP6-sufficient CD103+ B cells. Microarray analysis of NLRP6-sufficient and -deficient CD103+ B cells identified 79 significantly different genes including genes regulated by lipopolysaccharide (LPS), tretinoin, IL-10 and TGFb, which was confirmed in vitro following LPS stimulation. Furthermore, microbiota from NLRP6-deficient mice induced CD103+ B cells in colonized NLRP6-sufficient germ-free mice; however, the long-term maintenance of the CD103+ B cells required the absence of NLRP6 in the hosts, or continued exposure to microbiota from NLRP6-deficient mice. Discussion: Together, our data indicate that NLRP6 deficiency promotes expansion and maintenance of a novel TGF -dependent CD103+ Breg population. Thus, targeting NLRP6 therapeutically may prove clinically useful.

[Research progress on strain improvement of Acremonium chrysogenum by genetic engineering].

Acremonium chrysogenum, cephalosporin C (CPC) producing strain, is an important industrial microorganism. CPC is used to produce 7-ACA, a major intermediate for manufacturing of many first-line anti-infectious cephalosporin-antibiotics. The fermentation level of CPC determines the production, quality and cost of its downstream products. Therefore, it is necessary to develop the strains of A. chrysogenum. Along with the development of molecular biology, genetic manipulation technique is becoming more and more important in the field of molecular breeding. This paper reviews the latest research progresses on CPC biosynthesis and its regulation. Genetic manipulations of A. chrysogenum were summarized and concluded. We suggested that strain improvement of A. chrysogenum by means of induction and expression of biosynthetic and regulatory genes, as well as exogenous genes, and further optimization could be applied to different aspects including CPC production enhancement and metabolic pathway elongation, etc. Future direction of this field is also proposed. We believed that incorporation of comparative proteomics and genomic shuffling with molecular breeding could lead the achievements close to industry promptly.

A new approach to produce IgG4-like bispecific antibodies.

While achieving rapid developments in recent years, bispecific antibodies are still difficult to design and manufacture, due to mispair of both heavy and light chains. Here we report a novel technology to make bispecific molecules. The knob-into-hole method was used to pair two distinct heavy chains as a heterodimer. IgG4 S228P CH1-CL interface was then partially replaced by T-cell receptor α/ß constant domain to increase the efficiency of cognate heavy and light chain pairing. Following expression and purification, the bispecific antibody interface exchange was confirmed by Western blotting and LC-MS/MS. To ensure its validity, we combined a monovalent bispecific antibody against PD-1 (sequence from Pembrolizumab) and LAG3 (sequence from Relatlimab). The results showed that the molecule could be assembled correctly at a ratio of 95% in cells. In vitro functional assay demonstrated that the purified bispecific antibody exhibits an enhanced agonist activity compared to that of the parental antibodies. Low immunogenicity was predicted by an open-access software and ADA test.