Xenotransplantation has entered the clinical phase in an effort to address the global organ shortage. However, recent clinical studies have revealed that current xenografts from gene-edited (GE) pigs still pose a risk of immune rejection and biosafety concerns. In this study, we successfully produced a large batch of 582 GE cloned (GEC) pigs with 10-(GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hTBM/hCD39/hEPCR/hCD47) gene edits via gene editing and somatic cell cloning technologies, and successfully obtained the F1 generation. Phenotypic characterization of 10-GEC pigs revealed the deletion of three xenoantigens and the expression of seven human transgenes across various tissues. Digital droplet polymerase chain reaction and whole-genome sequencing revealed two copies of hCD46/hCD55/hCD59/hTBM/hCD39 and one copy of hEPCR/hCD47 in the pig genome with minimal off-target effects or damage to the porcine functional genes. The validation results showed that 10-GEC pigs could effectively inhibit attacks from human antibodies, complement and macrophages on porcine endothelial cells, and alleviated coagulation abnormalities between pigs and humans. Large-scale screening of pathogens revealed no evidence of 47 pathogens, including cytomegalovirus, in our 10-GEC pigs. Kidney, heart and liver xenografts from these 10-GEC pigs were transplanted into nonhuman primates (NHPs), which worked normally without hyperacute rejection (HAR). Among NHPs, the heart and liver orthotopic transplant recipients survived for 3 and 4 days, respectively, while the two kidney transplant recipients survived for 23 and 16 days, respectively. Pathological analysis showed interstitial hemorrhage and fibrosis, cellular hyperplasia with minor antibodies and complement deposition, but significantly reduced infiltration of CD68⁺ macrophages in 10-GEC pig kidney xenografts. In summary, we successfully produced specific pathogen-free 10-GEC donor pigs that resulted in effective mitigation of immune rejection upon multiorgan transplantation to NHPs.

Advancements in protein engineering have driven the continuous optimization of T-cell engagers (TCEs), resulting in remarkable clinical outcomes in the treatment of B-cell malignancies. Moreover, developing tri- or multispecific TCEs has emerged as a promising strategy to address the challenges of tumor heterogeneity and antigen escape. However, considerable obstacles remain, primarily in format design. In this study, we engineered BAFF-based TCEs with various formats that incorporate anti-CD3 Fab or IgG domains fused with BAFF ligands to target BAFF receptors (BAFFR, BCMA, and TACI). These constructs varied in valency and the presence or absence of long-acting elements such as Fc domains or the albumin binding domain consensus sequence (ABDCon). Although the inclusion of an Fc domain did not enhance sustained tumor eradication, variations in valency and spatial configuration profoundly influenced cytotoxicity. We identified TriBAFF/CD3/ABDCon as the optimal trifunctional construct, featuring an anti-CD3 Fab backbone with BAFF and ABDCon fused to the C-termini of the heavy and light chains. This design facilitates optimal immune synapse formation between the target cells and T cells and effectively controls tumor burdens in various B-cell malignancy models with good tolerability. Notably, TriBAFF/CD3/ABDCon outperformed conventional therapies, including blinatumomab and BAFF-based CAR-T cells, in models of heterogeneous leukemia and aggressive lymphoma. These findings underscore the potential of using natural ligands as antibody-targeting modules and provide valuable insights into the design of the next generation of multispecific TCEs, which hold promise for improving treatment outcomes in a wide range of malignancies and beyond.

GORK is a shaker-like potassium channel in plants that contains ankyrin (ANK) repeats. In guard cells, activation of GORK causes K⁺ efflux, reducing turgor pressure and closing stomata. However, how GORK is regulated remains largely elusive. Here, we solved the cryo-EM structure of Arabidopsis GORK, revealing an unusual symmetry reduction (from C4 to C2) feature within its tetrameric assembly. This symmetry reduction in GORK channel is driven by ANK dimerization, which disrupts the coupling between transmembrane helices and cytoplasmic domains, thus maintaining GORK in an autoinhibited state. Electrophysiological and structural analyses further confirmed that ANK dimerization inhibits GORK, and its removal restores C4 symmetry, converting GORK to an activatable state. This dynamic switching between C2 and C4 symmetry, mediated by ANK dimerization, presents a GORK target site that guard cells regulate to switch the plant K⁺ channel between inhibited and activatable states, thus controlling stomatal movement in response to environmental stimuli.