Endocytosis and lysosomal degradation are critical pathways that determine the intracellular trafficking and therapeutic efficacy of antibody-drug conjugates (ADCs). However, inefficient internalization and lysosomal trafficking often limit ADC potency. Here, we introduce receptor-ubiquitination-targeting ADCs (ubitaADCs), a class of ADCs engineered to simultaneously bind target receptors and E3 ubiquitin ligases, thereby inducing receptor ubiquitination to enhance endocytosis and lysosomal delivery. Using engineered ubitaADC targeting epidermal growth factor receptor (EGFR), we demonstrate that promoting receptor ubiquitination accelerates internalization and lysosomal trafficking, leading to enhanced intracellular drug release and improved tumor cell killing. Mechanistic studies reveal that E3 ligase recruitment facilitates receptor ubiquitination, triggering endocytosis and subsequent lysosomal degradation. In vivo, ubitaADCs exhibit superior antitumor efficacy compared to conventional ADCs. This study establishes receptor ubiquitination as a powerful strategy to optimize ADC function and provides a generalizable approach for improving targeted protein degradation in therapeutic applications.

Recurrent missense mutations in the human epidermal growth factor receptor 2 (HER2) have been identified across various human cancers. Among these mutations, the active S310F mutation in the HER2 extracellular domain stands out as not only oncogenic but also confers resistance to pertuzumab, an antibody drug widely used in clinical cancer therapy, by impeding its binding. In this study, we have successfully employed computational- aided rational design to undertake directed evolution of pertuzumab, resulting in the creation of an evolved pertuzumab variant named Ptz- SA. This variant, with only two mutations (T30S/D31A) located on its heavy chain, effectively reinstates binding to the mutated antigen, at the expense of a 35- fold reduction in binding affinity potent killing capacity through antigen- dependent cytotoxicity. Moreover, upon engineering Ptz- SA into antibody-drug conjugates, such as Ptz- SA- MMAE, it manifests notable in vitro and in vivo antitumor efficacy by efficiently delivering cytotoxic payload into tumor cells expressing HER2 (S310F). Cryoelectron microscopy studies elucidate the molecular mechanism underlying the restored binding ability of Ptz- SA toward the S310F mutation. The steric hindrance induced by the S310F mutation is efficiently circumvented by the T30S and D31A mutations, which provides adequate space to accommodate the larger phenylalanine. Additionally, Ptz- SA also exhibits binding capacity to HER2 (S310Y), another mutation occurring at the S310 site of HER2 with high frequency. The computational- aided evolution of pertuzumab provides an alternative strategy for overcoming point mutation- mediated resistance to therapeutic antibodies.

Presentation of cases Dr.Ye(Pulmonologist):A seven-year-old boy was admitted to this hospital due to persistent cough. The patient was in good health until two weeks before the current presentation,when he developed a mild,non-pro-ductive cough without wheezing,shortness of breath,chest pain,or hemoptysis.Initially,his parents did not take him to the hospital and administered oral cefixime and montelukast for four days without improvement.Then chest computed tomography(CT)scan at a local hospital revealed bilat-eral pulmonary inflammation with left-lung hyperlucency(Fig.1a,b).

Thyroid dysfunction is associated with the risk of cardiovascular disease; however, whether plasma thyroid-stimulating hormone (TSH) levels in subjects with euthyroidism affect the risk of cardiovascular disease remains unclear. This study aimed to investigate the causal association between plasma TSH levels and cardiovascular diseases, particularly ischemic heart disease and heart failure (HF). Summary statistics from the Integrative Epidemiology Unit Open genome-wide association studies Project and FinnGen consortium were used to investigate the causal relationship between plasma TSH levels and the risk of cardiovascular diseases. Two-sample Mendelian randomization analysis using inverse-variance weighting as the primary method was performed. The MR Pleiotropy RESidual Sum and Outlier and leave-one-out methods were used to ensure the robustness of our findings. Genetically determined plasma TSH levels were associated with major coronary heart disease events (OR 1.0557, 95% CI 1.0141–1.0991), all-cause HF (OR 0.9587, 95% CI 0.9231–0.9956), and HF + non-ischemic cardiomyopathy (OR 0.9318, 95% CI 0.8786–0.9882). After the Bonferroni correction, the causation described above disappeared. In the secondary analysis, genetically determined higher TSH levels were associated with a higher risk for unstable angina pectoris (OR 1.0913, 95% CI 1.0350–1.1507), but were associated with a lower risk for HF + overweight (OR 0.9265, 95% CI 0.8821–0.9731). These results were further validated using sensitivity analysis. Our findings show that increased plasma TSH levels in patients with euthyroidism may increase the risk of unstable angina pectoris but reduce the risk of HF in overweight patients. This evidence indicates that plasma TSH levels may need to be carefully controlled in specific patients.

Antibody-based bispecific T cell engagers (TCEs) that redirect T cells to kill tumor cells have shown a promising therapeutic effect on hematologic malignancies. However, tumor-specific targeting is still a challenge for TCEs, impeding the development of TCEs for solid tumor therapy. The major histocompatibility complex (MHC) presents almost all intracellular peptides (including tumor-specific peptides) on the cell surface to be scanned by the TCR on T cells. With the premise of choosing optimal peptides, the final complex peptide–MHC could be the tumor-specific target for TCEs. Here, a novel TCR-directed format of a TCE targeting peptide–MHC was designed named IgG-T-TCE, which was modified from the IgG backbone and prepared in a mammalian cell expression system. The recombinant IgG-T-TCE-NY targeting NY-ESO-1157–165/HLA-A*02:01 could be generated in HEK293 cells with a glycosylated TCR and showed potency in T cell activation and redirecting T cells to specifically kill target tumor cells. We also found that the in vitro activity of IgG-T-TCE-NY could be leveraged by various anti-CD3 antibodies and Fc silencing. The IgG-T-TCE-NY efficiently inhibited tumor growth in a tumor–PBMC co-engrafted mouse model without any obvious toxicities.

Inadequate antigen-specific T cells activation hampers immunotherapy due to complex antigen presentation. In addition, therapeutic in vivo T cell expansion is constrained by slow expansion rates and limited functionality. Herein, we introduce a model fusion protein termed antigen-presenting cell-mimic fusion protein (APC-mimic), designed to greatly mimicking the natural antigen presentation pattern of antigen-presenting cells and directly expand T cells both in vitro and in vivo. The APC-mimic comprises the cognate peptide-human leukocyte antigen (pHLA) complex and the co-stimulatory marker CD80, which are natural ligands on APCs. Following a single stimulation, APC-mimic leads to an approximately 400-fold increase in the polyclonal expansion of antigen-specific T cells compared with the untreated group in vitro without the requirement for specialized antigen-presenting cells. Through the combination of single-cell TCR sequencing (scTCR-seq) and single-cell RNA sequencing (scRNA-seq), we identify an approximately 600-fold monoclonal expansion clonotype among these polyclonal clonotypes. It also exhibits suitability for in vivo applications confirmed in the OT-1 mouse model. Furthermore, T cells expanded by APC-mimic effectively inhibits tumor growth in adoptive cell transfer (ACT) murine models. These findings pave the way for the versatile APC-mimic platform for personalized therapeutics, enabling direct expansion of polyfunctional antigen-specific T cell subsets in vitro and in vivo.

Antibody-drug conjugates (ADCs) achieve therapeutic effects through toxin delivery inside cells, targeting highabundance antigens. However, tumor heterogeneity and the low abundance of tumor-specific antigens underscore the urgent requirement for developing a flexible, multifunctional, and high-capacity drug-delivery platform. The current study developed a self-assembled ADC platform called the high drug-antibody-ratio ADC (HDADC). Cytotoxic payloads were efficiently conjugated into a 12-arm deoxyribonucleic acid (DNA) branched junction via DNA-mediated precise self-assembly to achieve a high drug-antibody ratio (DAR). Anti-EGFR HDADC showed strong efficacy in causing cytotoxicity and suppressing tumor growth in an A431 xenograft mouse model. Furthermore, the Cy5-conjugated HD-ADC platform was a simple and effective method for improving fluorescent signal detection, enabling the detection of targets-such as neoantigens-with ultralow-expression levels. The HD-ADC platform supports the assembly of various functional components, providing the foundation for usage across multiple antibody-mediated targeted therapies and diagnostics.

X-linked hyper-IgM (X-HIGM), which results from mutations of the CD40 ligand gene (CD40LG) located on chromosome Xq26.3, is characterized by a defective T-B lymphocyte cross talk and class switch recombination (CSR). The present study aimed to evaluate the expression of CD40L and lymphocyte subsets using flow cytometry and to identify the novel genetic defect of CD40LG responsible for X-HIGM in a Chinese family. We reported an X-HIGM case caused by a novel mutation in CD40LG. The expression of CD40L was absent on the surface of activated CD4+ T cells evaluated using flow cytometry. The total number of mature B cells in circulation was normal, but memory B cells were significantly decreased. In helper T cells, Th2 was dominant, and the numbers of Th1 and Th17 were decreased. The results of genetic analysis revealed a new causative mutation in CD40L (NM_000074;exon5;c.505_506del), which leads to a change in amino acids (p.Y169Lfs*31) appearing in the proband. The frame shift mutation led to incorrect amino acid translation and loss of β-pleated sheet and loop region, which produced a mutant dysfunctional protein. This study provides a complete picture of X-HIGM and broadens our knowledge of the pathogenicity of the CD40L variant spectrum.

Targeting single tumor antigens makes it difficult to provide sufficient tumor selectivity for T cell engagers (TCEs), leading to undesirable toxicity and even treatment failure, which is particularly serious in solid tumors. Here, we designed novel trispecific TCEs (TriTCEs) to improve the tumor selectivity of TCEs by logic-gated dual tumor-targeting. TriTCE can effectively redirect and activate T cells to kill tumor cells (∼18 pM EC50) by inducing the aggregation of dual tumor antigens, which was ∼70- or 750- fold more effective than the single tumor-targeted isotype controls, respectively. Further in vivo experiments indicated that TriTCE has the ability to accumulate in tumor tissue and can induce circulating T cells to infiltrate into tumor sites. Hence, TriTCE showed a stronger tumor growth inhibition ability and significantly prolonged the survival time of the mice. Finally, we revealed that this concept of logic-gated dual tumor-targeted TriTCE can be applied to target different tumor antigens. Cumulatively, we reported novel dual tumor-targeted TriTCEs that can mediate a robust T cell response by simultaneous recognition of dual tumor antigens at the same cell surface. TriTCEs allow better selective T cell activity on tumor cells, resulting in safer TCE treatment.

Inadequate T cell activation has severely limited the success of T cell engager (TCE) therapy, especially in solid tumors. Enhancing T cell activity while maintaining the tumor specificity of TCEs is the key to improving their clinical efficacy. However, currently, there needs to be more effective strategies in clinical practice. Here, we design novel superantigen-fused TCEs that display robust tumor antigen-mediated T cell activation effects. These innovative drugs are not only armed with the powerful T cell activation ability of superantigens but also retain the dependence of TCEs on tumor antigens, realizing the ingenious combination of the advantages of two existing drugs. Superantigen-fused TCEs have been preliminarily proven to have good (>30-fold more potent) and specific (>25-fold more potent) antitumor activity in vitro and in vivo. Surprisingly, they can also induce the activation of T cell chemotaxis signals, which may promote T cell infiltration and further provide an additional guarantee for improving TCE efficacy in solid tumors. Overall, this proof-of-concept provides a potential strategy for improving the clinical efficacy of TCEs.

Acetaminophen (APAP), a common antipyretic and analgesic drug, is considered the most common cause of drug-induced liver injury (DILI). The mechanism of liver injury induced by APAP is mainly related to oxidative stress and inflammatory reaction. Herein, we report a simple and efficient one-step synthesis of Prussian blue (PB) nanozymes with multiple antioxidant enzymatic activities that effectively treat APAP-induced DILI. At the cellular level, reaching 10 mu g/mL PB nanozymes can effectively scavenge intracellular reactive oxygen species (ROS), reduce mitochondrial membrane potential drop, and inhibit hepatocyte apoptosis. According to in vivo experimental studies, the levels of serum biochemical indicators and histopathological examination of DILI mice livers showed that 12.5 mg/kg PB nanozymes could effectively inhibit liver necrosis and 25 mg/kg PB nanozymes achieved the same therapeutic effect as 300 mg/kg NAC. More importantly, compared with NAC, PB nanozymes can still attenuate APAP-induced acute liver injury in mice after APAP-induced acute liver injury in mice for 3 h. Therefore, PB nanozymes can effectively prolong the therapeutic time window, revealing the potential of PB nanozymes in clinical applications for advanced DILI treatment. Furthermore, the therapeutic mechanism studies have shown that PB nanozymes with abundant and variable valence states could not only directly scavenge ROS but also through the Keap1-Nrf2/HO-1 pathway to reduce oxidative stress. Moreover, the decreased expression levels of myeloperoxidase and F4/80 in the liver, which are markers of neutrophil and macrophage infiltration, indicated that the Prussian blue nanozymes modulates inflammation to protect against APAP-induced acute liver injury. Consequently, our findings suggested that PB nanozymes have excellent clinical application prospects for acetaminophen-induced acute liver injury.

Monoclonal antibodies constitute a promising class of targeted anticancer agents that enhance natural immune system functions to suppress cancer cell activity and eliminate cancer cells. The successful application of IgG monoclonal antibodies has inspired the development of various types of therapeutic antibodies, such as antibody fragments, bispecific antibodies, and antibody derivatives (e.g., antibody-drug conjugates and immunocytokines). The miniaturization and multifunctionalization of antibodies are flexible and viable strategies for diagnosing or treating malignant tumors in a complex tumor environment. In this review, we summarize antibodies of various molecular types, antibody applications in cancer therapy, and details of clinical study advances. We also discuss the rationale and mechanism of action of various antibody formats, including antibody-drug conjugates, antibody-oligonucleotide conjugates, bispecific/multispecific antibodies, immunocytokines, antibody fragments, and scaffold proteins. With advances in modern biotechnology, well-designed novel antibodies are finally paving the way for successful treatments of various cancers, including precise tumor immunotherapy, in the clinic.

Axillary lymph node dissection (ALND) can be omitted in the part of the breast-conserving patients with positive sentinel lymph nodes (SLNs) since Z0011 trial has presented. Nevertheless, to date, no studies revealed the influence of different tracing modalities (single tracer versus dual tracers) for sentinel lymph node biopsy (SLNB) on axillary management referring to Z0011 trial criteria. This study aimed to assess whether different tracing modalities of SLNB have impact on axillary management referring to Z0011 trial criteria. The clinical data of breast-conserving patients who underwent SLNB guided by combination of methylene blue (MB) and indocyanine green(ICG) were retrospectively analyzed in our center. The numbers of metastatic (positive) SLNs guided by the single tracer and the dual tracer were compared by self-control study. 127 patients with 1–2 metastatic SLNs dyed by MB [(recorded as MB(+))]were retrieved from our database between 2016 and 2020. In these cases, 53 patients contained 86 SLNs, which were ICG staining but MB negative staining (recorded as ICG(+)/MB(−)). In addition, 16 patients contained 20 metastatic SLNs with ICG(+)/MB(−). There were six patients finally excluded patients (6/127, 4.7%) who initially met the criteria of Z0011 trial, because the further detection of ICG(+)/MB(−) SLNs led the total numbers of positive SLNs over two. The difference was statistically significant. Single tracing modality may underestimate the positive SLN numbers compared to dual tracing modality. Different tracing modalities of SLNB will significantly affect Axillary management referring to Z0011 trial criteria.

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFR S492R - or EGFR G465R -driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFR S492R , which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFR G465R ). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFR S492R and EGFR G465R as well as EGFR WT . The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.

目的:探讨同伴互助教学(Peer-Assisted Learning,PAL)在儿科见习临床技能中的效果.方法:选择浙江大学医学院2015级本科见习生146名作为研究对象,实验组(n=73)采用PAL法,对照组(n=73)采用传统教学法,比较两组的教学效果.结果:临床技能操作成绩两组间无差异(P>0.05),但实验组教师授课时间明显减少;问卷调查显示学生普遍认可PAL法.结论:在儿科见习临床技能中实施PAL,即能保证学习效果又能减轻临床教师的工作负担,值得推广应用.

Bispecific antibodies (BsAbs) for T cell engagement have shown great promise in cancer immunotherapy, and their clinical applications have been proven in treating hematological malignance. Bispecific antibody binding fragment (BiFab) represents a promising platform for generating non-Fc bispecific antibodies. However, the generation of BiFab is still challenging, especially by means of chemical conjugation. More conjugation strategies, e.g., enzymatic conjugation and modular BiFab preparation, are needed to improve the robustness and flexibility of BiFab preparation. We successfully used chemo-enzymatic conjugation approach to generate bispecific antibody (i.e., BiFab) with Fabs from full-length antibodies. Paired click handles (e.g., N3 and DBCO) was introduced to the C-terminal LPETG tag of Fabs via sortase A mediated transpeptidation, followed by site-specific conjugation between two click handle-modified Fabs for BiFab generation. Both BiFabCD20/CD3 (EC50 = 0.26 ng/mL) and BiFabHer2/CD3 exhibited superior efficacy in mediating T cells, from either PBMC or ATC, to kill target tumor cell lines while spared antigen-negative tumor cells in vitro. The BiFabCD20/CD3 also efficiently inhibited CD20-positive tumor growth in mouse xenograft model. We have established a facile sortase A-mediated click handle installation to generate homogeneous and functional BiFabs. The exemplary BiFabs against different targets showed superior efficacy in redirecting and activating T cells to specifically kill target tumor cells, demonstrating the robustness of sortase A-mediated “bio-click” chemistry in generating various potent BiFabs. This approach also holds promise for further efficient construction of a Fab derivative library for personalized tumor immunotherapy in the future.

Neoantigen-based personalized vaccination has emerged as a viable method for tumor immunotherapy. Here we set up a DNA-based neoantigen vaccine platform with comprehensive identification of individual somatic mutations using whole-exome sequencing (WES) and RNA-seq, bioinformatic prediction of neo-epitopes, dendritic cell (DC)-based efficacy prevalidation of vaccine candidates, optimization of the DNA vaccine and its nanocarrier and adjuvant, and preparation of a liposome-encapsulated multiepitope DNA vaccine. The DNA vaccine was efficiently uptaken by DCs and induced effective immune response against mouse melanoma cells, leading to significant inhibition of melanoma tumor growth and reduction of lung metastasis in a mouse model. Numerous intratumoral infiltrated CD8+ T-cells with specific in vitro killing ability towards melanoma cells were identified. Our study offers evidence that a multiepitope neoantigen DNA vaccine in a nanocarrier can be exploited for personalized tumor immunotherapy and as a reliable prevalidation approach for rapid enrichment of effective neoantigens.

The specific recognition of T cell receptors (TCR) and peptides presented by human leukocyte antigens (pHLAs) is the core step for T cell triggering to execute anti-tumor activity. However, TCR assembly and soluble expression are challenging, which precludes the broad use of TCR in tumor therapy. Herein, we used heterodimeric Fc to assist in the correct assembly of TCRs to achieve the stable and soluble expression of several TCRs in mammalian cells, and the soluble TCRs enable us to yield novel bispecific T cell engagers (TCR/aCD3) through pairing them with an anti-CD3 antibody. The NY-ESO-1/LAGE-1 targeted TCR/aCD3 (NY-TCR/aCD3) that we generated can redirect naïve T cells to specific lysis antigen-positive tumor cells, but the potency of the NY-TCR/aCD3 was disappointing. Furthermore, we found that the activation of T cells by NY-TCR/aCD3 was mild and unabiding, and the activity of NY-TCR/aCD3 could be significantly improved when we replaced naïve T cells with pre-activated T cells. Therefore, we employed the robust T cell activation ability of staphylococcal enterotoxin C2 (SEC2) to optimize the activity of NY-TCR/aCD3. Moreover, we found that the secretions of SEC2-activated T cells can promote HLA-I expression and thus increase target levels, which may further contribute to improving the activity of NY-TCR/aCD3. Our study described novel strategies for soluble TCR expression, and the optimization of the generation and potency of TCR/aCD3 provided a representative for us to fully exploit TCRs for the precision targeting of cancers.

Dear Editor, The epidermal growth factor receptor(EGFR),one member of the ErbB family of receptor tyrosine kinases,has been implicated in various epithelial malignancies.As a clinically validated target,both small-molecule tyrosine kinase inhibitors and antibody-based drugs have been approved by regulatory agencies.Anti-EGFR antibody-drug conjugates(ADCs)could kill target tumor cells irrespective of EGFR signaling.