We designed and developed 9MW2821, an anti-Nectin-4 antibody-drug conjugate (ADC) with an enzymatically cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) as the payload. Four bioanalytical assays for total antibodies, conjugated antibodies, conjugated payload, and free payload were then developed and validated for the comprehensive evaluation of the multiple drug forms of 9MW2821. Specific sandwich enzyme-linked immunosorbent assays were used to quantify total antibodies and conjugated antibody, showing good drug-to-antibody ratio (DAR) tolerance. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine free MMAE, and conjugated MMAE was quantified using a combination of ligand-binding assay (LBA) and LC-MS/MS. Based on these four assays, we studied the serum stability and monkey pharmacokinetic profiles of 9MW2821, and the in vivo DAR of 9MW2821 was calculated and dynamically monitored. In conclusion, we developed and validated series of bioanalytical assays to quantify multiple forms of 9MW2821, a new ADC, and used the assays to evaluate the serum stability and monkey pharmacokinetic characteristics. The results indicate good linker stability and suggest that the developed assays can be further used in clinical settings.

We designed and developed a novel DNA topoisomerase I inhibitor MF-6, which was a more potent cytotoxin and a more potent inducer of immunogenic cell death compared with DXd. To utilize MF-6's ability to induce antitumor immunity, a human epidermal growth factor receptor 2 (HER2)-targeted antibody-drug conjugate (ADC) trastuzumab-L6 that included a cleavable linker and MF-6 was developed. Different from traditional cytotoxic ADC, the antitumor activity of trastuzumab-L6 was assessed by inducing tumor cell immunogenic cell death, activating dendritic cells and cytotoxic CD8+ T cells to acquire durable adaptive immune memory. Tumor cells treated with trastuzumab-L6 were committed to immunogenic cell death, with upregulation of damage-associated molecular patterns and antigen presentation molecules. In a syngeneic tumor model with a mouse cell line that expressed human HER2, immunocompetent mice showed greater antitumor efficacy compared with nude mice. The trastuzumab-L6-cured immunocompetent mice acquired adaptive antitumor memory and rejected subsequent tumor cell challenge. The trastuzumab-L6 efficacy was abrogated when cytotoxic CD8+ T cells were depleted and enhanced when regulatory CD4+ T cells were depleted. The combination of trastuzumab-L6 with immune checkpoint inhibitors significantly increased antitumor efficacy. Enhanced T cell infiltration, dendritic cell activation, and decreased type M2 macrophages in tumor post trastuzumab-L6 administration confirmed the immune-activating responses. In conclusion, trastuzumab-L6 was considered to be an immunostimulatory agent, rather than a traditional cytotoxic ADC, and its antitumor efficacy was enhanced when combined with an anti-PD-L1 and anti-CTLA-4 antibody, which suggested a potential therapeutic strategy.

Trophoblast cell surface antigen 2 (Trop-2) is a kind of transmembrane glycoprotein on the cell surface, which is highly expressed in various tumors (breast cancer, non-small cell lung cancer, pancreatic cancer, gastric cancer, colon cancer, e. g.). Its high expression is associated with poor prognosis. As a popular target for tumor therapy, many Trop-2-targeted ADC drugs have been developed at home and abroad, one of which has successfully marketed. This paper reviews the research progress of the drugs at home and abroad.

TMAB001 is a humanized rabbit monoclonal antibody (mAb) designed to bind and neutralize human vascular endothelial growth factor (VEGF)-165. The purpose of the study was to investigate the pharmacokinetics (PK) and ocular tissue distribution after a single intravitreal (IVT) dose in rabbits and monkeys. Rabbits (2.5 mg/eye; n= 40) and monkeys (2.5 mg/eye; n= 12) received TMAB001 as a bilateral IVT dose. TMAB001 concentrations were measured in ocular tissues in all rabbits and monkeys by enzyme-linked immunosorbent assay (ELISA). TMAB001 and VEGF concentrations were measured in serum of monkeys by ELISA. Following a single bilateral IVT injection of TMAB001 2.5 mg/eye, the highest concentration was in vitreous humor, followed by retina and choroid, and the lowest concentration was in lens. In rabbits, TMAB001 was still detectable in ocular tissues at day 21 after single IVT dose, with the highest level in the vitreous humor and then retina, with longest t(1/2) in aqueous humor and shortest t(1/2) in choroid. In monkeys, t(max) in serum was 43 h and t(1/2) was approximately 5.5 days. C-max] in serum was much lower than that in vitreous, nearly 1/200. After IVT injection of TMAB001, total VEGF concentrations in serum and ocular tissues increased over time. VEGF concentration in retina and choroid increased over time, up to 336 h after administration. This study demonstrated that TMAB001 could reach the drug target sites-retina and choroid after a single bilateral IVT administration in rabbits and monkeys, with a long t(1/2) in vitreous humor. TMAB001 also showed strong capability to neutralize VEGF. The study further confirmed that full-length antibodies can also efficiently diffuse and distribute in ocular tissues.

AIMS:GLP-1-based strategies have many advantages in treatment of type 2 diabetes mellitus (T2DM), but native GLP-1 has a short half-life in the circulation, which limits its clinical application. The purpose of this study was to evaluate the effects of GW002, a novel recombinant GLP-1 analog fusion protein produced by linking the human GLP-1 analog C-terminus to the N-terminus of human serum albumin via a linker, in vitro and in BKS-db mice.METHODS:To determine whether GW002 can activate the GLP-1 receptor in cells, the level of luciferase expression was evaluated in vitro. In vivo, body weight, food intake, non-fasting and fasting blood glucose, oral glucose tolerance test, blood glucose and insulin levels, liver histology, liver function parameters and antibody levels in BKS-db mice were investigated to evaluate the effects of GW002. Albiglutide was chosen as a positive comparator.RESULTS:Cyclic adenosine monophosphate levels were increased in a dose-dependent manner in cells. In vivo studies demonstrated that GW002 lowers non-fasting and fasting blood glucose levels and improves glucose tolerance and insulin secretion in BKS-db mice. The degree of hepatic steatosis and hepatic biochemical indexes was also decreased. In this study, the mice body weight was not reduced significantly.CONCLUSIONS:The above results showed that the efficacy of GW002 in BKS-db mice displayed a significant hypoglycemic effect, which indicated that GW002 might be a potential candidate for the treatment of T2DM.