Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy, characterized by near-universal oncogenic KRAS mutations (most frequently the G12D variant) and profound apoptosis resistance. This creates critical therapeutic challenge: KRAS signaling not only drives tumor progression but also confers resistance apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To address this dual impediment, we developed a biomimetic co-delivery platform termed T-CNV:si, comprising TRAIL displaying cell-derived nanovesicles (CNVs) encapsulating KRASG12D-siRNA. This system enables tumor specific targeting via TRAIL while simultaneously silencing the core oncogene and dismantling TRAIL resistance. Mechanistically, we demonstrate that combined KRAS silencing and TRAIL-mediated apoptosis induction synergistically provoke severe endoplasmic reticulum (ER) stress, initiating a DDIT3-death receptor (DR) 4/5 amplification loop that reprograms tumor cells toward TRAIL hypersensitivity. In both cell-derived and patient derived xenograft models of KRASG12D-mutant PDAC, systemic administration of T-CNV:si achieved profound tumor suppression with an excellent safety profile. This work unveils a novel strategy that converts a KRASdriven resistance mechanism into a targetable vulnerability, offering a promising paradigm for the treatment of KRAS-mutant cancers.

ABO-incompatible kidney transplantation is widely used to meet the escalating need for organs. Current recipient-centric desensitization protocols involving antibody depletion through plasmapheresis increase the risk of infections, perioperative bleeding events and costs. Here we present a donor-centric desensitization protocol, converting type-A kidneys into enzyme-converted O kidneys during hypothermic perfusion to remove the A antigen from the kidneys. An ex vivo model resulted in no antibody-mediated injury. Encouraged by this, an enzyme-converted O kidney was transplanted into a type-O brain-dead recipient with a high titre of anti-A antibody, and no hyperacute rejection was observed. The graft was well tolerated with no evidence of antibody-mediated rejection for 2 days. Antibody-mediated lesions and complement deposition were found starting 3 days post-transplant, coinciding with A-antigen regeneration, and later higher Banff scores, suggesting an immune-mediated response. Single-cell sequencing confirms the elevated expression of accommodation-related genes, suggesting the potential for longer-term tolerance. This study provides a donor-centric organ engineering strategy and has the potential to broaden the reach of ABO-incompatible kidney transplantation, improving the fairness of and access to organ allocation. An ex vivo model and pre-clinical study in a brain-dead recipient provide enzyme-converted O organs to avoid hyperacute rejection in ABO-incompatible kidney transplant patients.

Tumor-targeting drug delivery holds great promise for cancer treatment but faces significant challenges in penetrating solid tumors to achieve optimal therapeutic efficacy. By harnessing the natural tissue-penetration effect conferred by the CendR motif, we identified that the nucleolin (NCL)-targeted peptide F3 possesses tumor-penetrating capabilities. Co-administration of F3 with doxorubicin and the apoptosis-inducing protein TRAIL enhanced effective tumor penetration and improved antitumor activity. Taking advantage of TRAIL's natural self-trimerization, we developed a novel fusion protein, F3-TRAIL. This design enabled the trivalent assembly of F3 when fused with TRAIL, significantly enhancing its binding to NCL-positive tumor endothelial and parenchymal cells, resulting in deeper tumor penetration and superior antitumor effects compared to TRAIL alone. Mechanistic studies revealed that the multivalent F3-enhanced engagement with tumor cells potentiated TRAIL to trigger death receptor-dependent apoptosis signaling, even in TRAIL-resistant tumor cells. Building on this success, we constructed F3-HexaTR using the SpyCatcher/SpyTag superglue ligation system to generate a hexameric TRAIL, further amplifying cytotoxicity and antitumor efficacy. Combined analysis of data from TCGA and GTEx revealed significantly elevated NCL expression across 18 solid tumor types, underscoring the clinical potential of F3-directed targeted therapy. These findings highlight that F3-mediated NCL targeting is an effective strategy to overcome tumor penetration barriers, particularly for protein drug delivery. This multivalent assembly approach represents an innovative avenue for enhancing the therapeutic efficacy of various agents in the treatment of solid tumors.

As T and NK cell exhaustion is attributed to increased expression of immune checkpoints and decreased production of proliferative cytokines by these cells, immune checkpoint-targeted delivery of proliferative cytokines might induce robust and sustained antitumor immune responses. Here, the expression profile of NKG2A was first found to be narrower than that of PD-1 in tumor-infiltrated immune cells. Moreover, unlike PD-1, NKG2A was predominantly co-expressed with IL-2Rβγ in tumor-infiltrated CD8+ T and NK cells, but not in Tregs, suggesting that NKG2A might be an ideal target for delivery of IL-2Rβγ agonists to overcome T and NK exhausting. For NKG2A-targeted delivery of an IL-2Rβγ agonist, a single molecule of de novo designed N215 endowed with Immunoglobin G(IgG)-binding ability was coupled to an antibody against NKG2A (αNKG2A) to produce αNKG2A-N215. NKG2A- and IL-2Rβγ-binding were well preserved in αNKG2A-N215, allowing αNKG2A-N215 to act as both an immune checkpoint inhibitor and a T and NK cell stimulator. Intravenously injected αNKG2A-N215 predominantly induced expansion of tumor-infiltrated CD8+ T and NK cells while showing little stimulation of Tregs. Compared with the separate combination using αNKG2A and N215, αNKG2A-N215 exerted a greater antitumor effect in mice bearing MC38 or B16/F1 tumors. 50% of mice bearing MC38 tumors were cured by αNKG2A-N215, and long-term immunological memory against the tumor was induced in these mice. These results indicate that NKG2A is another ideal target for delivery of an IL-2Rβγ agonist, and αNKG2A-N215, with specificities for both NKG2A and IL-2Rβγ, might be developed as a novel agent for immunotherapy.

Although non-immunoglobin scaffold binders with high affinity and broad spectrum for albumin are attractive for lab-scale albumin purification, affinity chromatography based on these binders has not been developed. Here, the albumin-binding capabilities of representative binders, including protein G-derived albumin binding domain (ABD), albumin binding nanofitins (ABNF), and human serum albumin affimer 31 (HSA31) were predicted by interaction structure analysis and verified by experimental assays. Interaction structure prediction suggested that ABD possessed great potential to bind human (HSA), rhesus monkey (RhSA), mouse (MSA), and rat serum albumin (RSA), whereas ABNF might only bind HSA and bovine serum albumin (BSA), and HSA31 might not bind any of the tested albumins. Subsequent experimental assays demonstrated that ABD was similar to ABNF in HSA and RhSA binding but was superior to ABNF in MSA and RSA binding. ABNF exhibited greater BSA-binding capability than that exhibited by ABD. Both ABD and ABNF showed little binding to rabbit serum albumin (RbSA) and HSA31 showed no binding to all tested albumins. ABD and ABNF could be conjugated to prepackaged NHS-activated HP columns at similar ligand densities. For purification of albumin from plasma, the yields of ABD-HP column for HSA, RhSA, MSA, and RSA were approximately 20 mg, which was 3-4 times higher than that of ABNF-HP column using either Gly-HCl or sodium caprylate as elution buffer. Notably, BSA could only be recovered from plasma using ABNF-HP column with a yield of 5-8 mg. However, little albumin could be purified using HSA31-HP column. These results indicate that both ABD- and ABNF-HP columns could be used as novel tools for purification of broad-spectrum albumins.

Pancreatic cancer is a malignant solid tumor that contains a significant number of cancer-associated fibroblasts (CAFs). Clinical trials have confirmed that CAF-targeted radionuclide therapy can suppress tumor growth and extend the survival of patients; therefore, quantifying CAFs by molecular imaging of CAF biomarkers is helpful for assessing disease progression and therapeutic responses of pancreatic cancer. In our previous study, we found that platelet-derived growth factor receptor beta (PDGFRβ) was highly expressed on various fibroblast cells, and a novel affibody (ZPDGFRβ) with highly specific binding to PDGFRβ had been developed. Herein, we verified the high expression of PDGFRβ on CAFs in pancreatic cancer tissues, and the ZPDGFRβ affibody was radiolabeled with 64Cu to obtain a [64Cu]Cu-NOTA-ZPDGFRβ conjugate with radiochemical purity higher than 95%. Biodistribution studies showed that tumor uptake of [64Cu]Cu-NOTA-ZPDGFRβ reached the peak of 7.28 ± 0.92 at 6 h postinjection, and the tumor-to-pancreas ratio continuously increased to reach the peak of 25.9 ± 8.18 at 24 h postinjection. Positron emission tomography (PET) imaging with [64Cu]Cu-NOTA-ZPDGFRβ showed ideal tumor uptake and imaging capability in mice bearing both subcutaneous xenografts and in situ grafts. Our results demonstrated that the [64Cu]Cu-NOTA-ZPDGFRβ conjugate could be applied as a promising PDGFRβ-targeted radiotracer for PET imaging of pancreatic cancer.

Modular assembly of multivalent therapeutics with precise modulation of pharmacokinetic and pharmacological properties remains a critical challenge in drug development. Here, we present ATPlug, a self-assembling protein platform that integrates three crucial functional modules: a trimerization domain to enhance avidity, a SpyCatcher module for efficient conjugation, and an albumin-binding domain to optimize pharmacokinetics and tissue selectivity. This rational design facilitates the modular assembly of multivalent artificial antibodies and antibody-drug conjugates (ADCs), demonstrating remarkable versatility through the successful incorporation of diverse therapeutic modules targeting epidermal growth factor receptor (EGFR), programmed cell death ligand 1 (PD-L1), and vascular endothelial growth factor (VEGF). The trivalent constructs exhibited up to 30-fold enhancement in target binding avidity and extended plasma half-life via endogenous albumin hitchhiking. Notably, the ATPlug-customized modular ADCs achieved binding affinities of 1.8 nM for EGFR and exhibited selective cytotoxicity toward EGFR-overexpressing tumor cells, resulting in potent tumor suppression efficacy. This plug-and-play strategy provides a framework for next-generation therapeutics combining customized multivalency with multidrug synergies.

Due to its tumor homing and long serum half-life, albumin is an ideal drug carrier for chemotherapy. For endogenous albumin hitchhiking with high cargo loading, a trimeric albumin-binding domain (ABD), i.e., ABD-Tri is designed by fusing an ABD with high specificity and affinity for albumin to a self-trimerizing domain (Tri) with an additional cysteine residue. ABD-Tri is highly (40 mg L-1) expressed as soluble and trimeric proteins in Escherichia coli (E. coli). Once mixed together, ABD-Tri rapidly and specifically forms a stable complex with albumin under physiological conditions without obviously changing its receptor- and cell-binding and tumor-homing properties. Maleimide-modified prodrugs are highly effectively conjugated to ABD-Tri to produce homogenous ABD-Tri-prodrugs with triple cargo loading under physiological conditions by thiol-maleimide click chemistry. Unlike the maleimide moiety, which can only mediate time- and concentration-dependent albumin binding, ABD-Tri mediated fast (within several minutes) albumin binding of drugs even at extremely low concentrations (µg mL-1). Compared to maleimide-modified prodrugs, ABD-Tri-prodrugs exhibit better tumor homing and greater in vivo antitumor effect, indicating that conjugation of chemical drug to ABD-Tri outperforms maleimide modification for endogenous albumin hitchhiking. The results demonstrate that ABD-Tri may serve as a novel platform to produce albumin-binding prodrugs with high cargo-loading capacity for tumor-targeted chemotherapy.

Purpose Noninvasive quantifying activated hepatic stellate cells (aHSCs) by molecular imaging is helpful for assessing disease progression and therapeutic responses of liver fibrosis. Our purpose is to develop platelet-derived growth factor receptor β (PDGFRβ)-targeted radioactive tracer for assessing liver fibrosis by positron emission tomography (PET) imaging of aHSCs. Methods Comparative transcriptomics, immunofluorescence staining and flow cytometry were used to evaluate PDGFRβ as biomarker for human aHSCs and determine the correlation of PDGFRβ with the severity of liver fibrosis. The high affinity affibody for PDGFRβ (Z PDGFRβ ) was labeled with gallium-68 ( 68 Ga) for PET imaging of mice with carbon tetrachloride (CCl 4 )-induced liver fibrosis. Binding of the [ 68 Ga]Ga-labeled Z PDGFRβ ([ 68 Ga]Ga-DOTA-Z PDGFRβ ) for aHSCs in human liver tissues was measured by autoradiography. Results PDGFRβ overexpressed in aHSCs was highly correlated with the severity of liver fibrosis in patients and CCl 4 -treated mice. The 68 Ga-labeled Z PDGFRβ affibody ([ 68 Ga]Ga-DOTA-Z PDGFRβ ) showed PDGFRβ-dependent binding to aHSCs. According to the PET imaging, hepatic uptake of [ 68 Ga]Ga-DOTA-Z PDGFRβ increased with the accumulation of aHSCs and collagens in the fibrotic livers of mice. In contrast, hepatic uptake of [ 68 Ga]Ga-DOTA-Z PDGFRβ decreased with spontaneous recovery or treatment of liver fibrosis, indicating that the progression and therapeutic responses of liver fibrosis in mice could be visualized by PDGFRβ-targeted PET imaging. [ 68 Ga]Ga-DOTA-Z PDGFRβ also bound human aHSCs and visualized fibrosis in patient-derived liver tissues. Conclusions PDGFRβ is a reliable biomarker for both human and mouse aHSCs. PDGFRβ-targeted PET imaging could be used for noninvasive monitoring of liver fibrosis in mice and has great potential for clinical translation.

Platelet-derived growth factor receptor β (PDGFRβ) has been demonstrated to be an effective biomarker for a variety of malignant cancers, and affibody-based PDGFRβ molecules have potential as positron emission tomography (PET) tracers for the diagnosis of cancers. Based on previous pharmacokinetics studies, short-lived positron emission radionuclides, such as fluorine-18 and gallium-68, would be more suitable for affibody-based PET imaging. Thus, in the present study, we prepared a gallium-68-labeled PDGFRβ-targeting dimeric affibody conjugate and evaluated its capability for visualizing malignant tumors by micro-PET/computed tomography (CT) imaging. The PDGFRβ-targeting ZPDGFRβ affibody was conjugated with the p-NCS-Bn-DOTA macrocyclic ligand and radiolabeled with gallium-68 to generate the 68Ga-DOTA-ZPDGFRβ PET probe . Then, several types of malignant carcinoma cells (U-87 MG, LS 174T, A549, H1688, and H446) were used to evaluate the targeted cellular binding capability of the PET probe through in vitro/in vivo cellular assays and whole-body imaging by micro-PET/CT. The 68Ga-DOTA-ZPDGFRβ was successfully prepared with a radiochemical yield of 93% and exhibited ideal stability for up to 4 h at room temperature in vitro. This radioactive conjugate demonstrated specific binding ability with PDGFRβ-expressing U-87 MG cells, which was suppressed by PDGFRβ ligands. The biodistribution of 68Ga-DOTA-ZPDGFRβ indicated fast liver clearance and a kidney-bladder excretion route. The U-87 MG xenografted tumor was clearly visualized with 68Ga-DOTA-ZPDGFRβ at 1 h postinjection using micro-PET/CT imaging. 68Ga-DOTA-ZPDGFRβ is a potential radiopharmaceutical for the diagnosis of PDGFRβ-expressing tumors.

Purpose Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel–targeted molecular imaging might be effective for early diagnosis of HCC. Herein, we developed a novel trimeric affibody (Z TRI ) with highly specific binding to the platelet-derived growth factor receptor beta (PDGFRβ). The aim of this study is to evaluate the feasibility of 68 Ga-radiolabeled Z TRI ([ 68 Ga]Ga-DOTA-Z TRI ) as PET tracer for diagnosis of HCC. Methods The bioinformatics analysis of clinical database and immunoblotting of clinical specimens were performed to validate the potential of PDGFRβ as HCC biomarker. The trimeric affibody Z TRI was conjugated with DOTA-NHS-ester and radiolabeled with 68 Ga to produce [ 68 Ga]Ga-DOTA-Z TRI conjugate. Immunoreactivity and specific uptake of [ 68 Ga]Ga-DOTA-Z TRI were assessed by dose-dependent cell binding, autoradiography, and biodistribution analysis. [ 68 Ga]Ga-DOTA-Z TRI PET/CT scanning of diethylnitrosamine (DEN)-induced primary HCC rats and a rare case of idiopathical HCC rhesus monkey was performed to evaluate the imaging capability and radiation dosimetry of [ 68 Ga]Ga-DOTA-Z TRI in vivo. Results Excessive PDGFRβ was validated as a representative biomarker of HCC neovascularization. The radiolabeling of [ 68 Ga]Ga-DOTA-Z TRI was achieved at more than 95% radiochemical yield. In vitro assays showed specific uptake of [ 68 Ga]Ga-DOTA-Z TRI in HCC tumor vessels by autoradiography. Animal PET/CT imaging with [ 68 Ga]Ga-DOTA-Z TRI successfully visualized the tumor lesions in primary HCC rats and rhesus monkey, and indicated radiation absorbed dose of 2.03E-02 mSv/MBq for each scanning. Conclusions Our results demonstrated that [ 68 Ga]Ga-DOTA-Z TRI conjugate could be applied as a promising PET tracer for early diagnosis of hepatocellular carcinoma.

Antibiotic resistance in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus remains a major public health concern worldwide. Furthermore, these microbes frequently co-exist in biofilm-associated infections, largely nullifying antibiotic-based therapy. Therefore, it is imperative to develop an efficient therapeutic strategy for combating infections caused by polymicrobial biofilms. In this study, we investigated the antibacterial and antibiofilm activity of the bacteriophage endolysin Ply113 in vitro. Ply113 exhibited high and rapid lytic activity against E. faecium, E. faecalis, and S. aureus, including vancomycin-resistant Enterococcus and methicillin-resistant S. aureus isolates. Transmission electron microscopy revealed that Ply113 treatment led to the detachment of bacterial cell walls and considerable cell lysis. Ply113 maintained stable lytic activity over a temperature range of 4–45°C, over a pH range of 5.0–8.0, and in the presence of 0–400 mM NaCl. Ply113 treatment effectively eliminated the mono-species biofilms formed by E. faecium, E. faecalis, and S. aureus in a dose-dependent manner. Ply113 was also able to eliminate the dual-species biofilms of E. faecium–S. aureus and E. faecalis–S. aureus. Additionally, Ply113 exerted potent antibacterial efficacy in vivo, distinctly decreasing the bacterial loads in a murine peritoneal septicemia model. Our findings suggest that the bacteriophage endolysin Ply113 is a promising antimicrobial agent for the treatment of polymicrobial infections.

Prompting higher-order death receptor (DR) clustering by increasing the valency of DR agonist is efficient to induce apoptosis of tumor cells. As an attractive DR agonist with superior biosafety, the trimeric tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts limited antitumor effect in patients, which is predomi-nantly attributed to its low DR clustering ability and short serum half-life. Previous antibody scaffolds-based engineering strategies to increase the valency and/or prolong the serum half-life of TRAIL improve apoptosis induction, however, often produce large proteins with poor tumor penetration. Covalent protein ligation mediated by small molecular superglues such as SpyTag/SpyCatcher might be a novel strategy to assemble higher-order TRAIL variants. Upon fusion to TRAIL promotor, SpyTag/SpyCatcher molecular superglue prefer-entially ligated two trimeric TRAIL to produce a hexameric TRAIL variant, HexaTR, exhibiting a significantly increased apoptosis induction. In addition, an albumin-binding HexaTR, ABD-HexaTR, with a prolonged serum half-life by binding to endogenous albumin was also produced using the same strategy. Compared to the trimeric TRAIL, the hexameric HexaTR and ABD-HexaTR showed 20-50 times greater in vivo antitumor effect, resulting in eradication of several types of large (150-300 mm3) tumor xenografts. Combination with bortezomib carried by liposome further improved the antitumor effects of the hexavalent HexaTR and ABD-HexaTR in refractory cancer. Our results indicate that the superglue-mediated higher-order assembly is promising to improve the DR clustering and proapoptotic signaling of TRAIL, showing great advantages in constructing the next generation of DR agonists for cancer therapy.

Clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly limited by its inefficient apoptosis induction in tumor cells, which might be improved by using molecular superglue-mediated hyperoligomerization to increase its valency. Here, the minimal superglue peptide pairs, including Snoopligase-catalyzed SnoopTagJr/SnoopDogTag and SpyStapler-catalyzed SpyTag/SpyBDTag, were individually fused at the N- or C-terminus of the TRAIL promoter to produce superglue-fusion TRAIL variants. Similar to native trivalent TRAIL, these superglue-fusion TRAIL variants were highly expressed in Escherichia coli (E. coli) and spontaneously trimerized. In the presence of Snoopligase or SpyStapler, the trivalent superglue-fusion TRAIL variants were predominantly crosslinked into hexavalent TRAIL variants. Nevertheless, Snoopligase was more efficient than SpyStapler in the production of hexavalent TRAIL variants. In particular, Snoopligase-catalyzed trivalent TRAIL variants with N-terminal fusion of SnoopTagJr/SnoopDogTag produced hexavalent SnHexaTR with the highest yield (∼70%). The in vitro cytotoxicity of SnHexaTR was 10-40 times greater than that of TRAIL in several tumor cells. In addition, compared to trivalent TRAIL, hexavalent SnHexaTR showed a longer serum half-life and greater tumor uptake, which resulted in eradication of 50% of tumor xenografts of TRAIL-sensitive COLO 205. In mice bearing TRAIL-resistant HT-29 tumor xenografts, hexavalent SnHexaTR combined with bortezomib encapsulated in liposomes also showed robust tumor growth suppression, indicating that hyperoligomerization mediated by minimal molecular superglue significantly increased the cytotoxicity and antitumor effect of TRAIL. As a novel anticancer agent candidate, the hexavalent SnHexaTR has great potential for clinical application in cancer therapy.

Multidrug resistance (MDR), which is common in colorectal cancer (CRC), induces high mortality in patients. Due to its robust and selective apoptosis induction in some CRC cells with MDR, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is attractive as a novel tool for CRC therapy. However, TRAIL is limited by its poor tumor-homing ability and inefficient apoptosis induction in CRC cells expressing low levels of death receptor (DR). Here, the tumor-homing RGR peptide (CRGRRST) was fused to TRAIL to produce RGR-TRAIL. Compared with TRAIL, RGR-TRAIL showed greater cell binding and cytotoxicity in CRC cells. In addition, RGR-TRAIL exerted significantly enhanced tumor uptake and growth suppression in mice bearing CRC tumor xenografts. Notably, RGR-TRAIL eradicated all tumor xenografts of DR-overexpressing COLO205 cells. However, TRAIL only showed mild tumor growth suppression under the same conditions, indicating that RGR fusion significantly increased the antitumor effect of TRAIL in DR-overexpressing CRC cells by improving tumor homing. Nevertheless, RGR fusion did not significantly enhance the antitumor effect of TRAIL in HT29 cells expressing low levels of DR. We found that DR expression in HT29 cells was enhanced by epidermal growth factor receptor (EGFR)-targeted photodynamic therapy (PDT). Moreover, both the in vitro and in vivo antitumor effects of RGR-TRAIL were significantly improved by combination with PDT. HT29 tumor xenografts (∼20%) were even eradicated by combination therapy. These results indicate that it is valuable to further evaluate the combination therapy of RGR-TRAIL and tumor-targeted PDT for clinical therapy of CRC with MDR.

Fragment antigen-binding (Fab) has several advantages in the treatment and diagnosis of some diseases. The lack of highly efficient affinity chromatography platform creates a purification bottleneck for the downstream processing of Fab-based products, which raises the urgent need for a novel immunoglobin G (IgG)-binding domain (IgBD) with both high affinity and broad specificity for Fab. SpGC3FabRR (designated CFab) was previously identified as a Fab-selective IgBD, which triggered our interest in evaluating the potential of CFab for Fab purification. However, we found that monomeric CFab showed weak Fab-binding. To increase its affinity, a self-trimerizing domain (tri) was fused to CFab to produce CFab-tri. It was found that CFab-tri existed as a trimer and showed promising binding to Fab derived from IgG of humans, rhesus monkeys, mice, rats, and rabbits. Affinity chromatography demonstrated that the recovery rates of Fab derived from IgG of humans, rats, mice, and rabbits by CFab-tri-HP column were 2- to 5-fold of those by protein G-HP column. Human Fab was effectively purified by both protein L- and CFab-tri-HP column. However, unlike CFab-tri-HP column, protein L-HP column was inefficient for purification of Fab derived from IgG of rats, mice, and rabbits. Notably, rat Fab spiked into the extract of Escherichia coli (E. coli) was effectively recovered by CFab-tri-HP column. These results indicate that CFab-tri outperforms protein G and protein L as a ligand for Fab purification, and CFab-tri-based affinity chromatography might be developed as a novel platform for Fab purification.

Chemotherapeutic multidrug resistance (MDR) is the major hindrance for clinical therapy of colorectal cancer (CRC). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with selective cytotoxicity might overcome MDR of CRC cells. Unfortunately, cross-resistance to TRAIL has been detected in many CRC cells, suggesting the need to combine TRAIL with sensitizers to combat refractory CRC. Our purpose is to explore the potential of combination therapy of TRAIL and tumor-cell targeted photodynamic therapy (PDT) in combating CRC with both chemotherapeutic MDR and TRAIL resistance. Methods: Tumor cell-targeted PDT was performed using a Ze-IR700 photosensitizer with high affinity for epidermal growth factor receptor (EGFR). The impact of PDT on the gene expression of CRC cells was revealed by RNA sequencing. The synergistic antitumor effect of long-acting TRAIL and PDT was evaluated in mice bearing tumor grafts of CRC cells with both chemotherapeutic MDR and TRAIL resistance. Results: Chemotherapeutic MDR and TRAIL resistance are common in CRC cells. Pretreatment of CRC cells with tumor cell-targeted PDT significantly (10-60 times) increased the sensitivity of these CRC cells to TRAIL by upregulating death receptors. Combination therapy, but not monotherapy, of long-acting TRAIL and PDT greatly induced apoptosis of CRC cells, thus efficiently eradicated large (~150 mm3) CRC tumor xenografts in mice. Conclusions: Tumor cell-targeted PDT extensively sensitizes CRC cells to TRAIL. Combination therapy of long-acting TRAIL and PDT is promising to combat CRC with both chemotherapeutic MDR and TRAIL resistance, which might be developed as a novel strategy for precision therapy of refractory CRC.

目的 制备血小板衍生生长因子受体β(platelet-derived growth factor receptor β,PDGFRβ)靶向性分子探针,并评价其用于肺癌光学分子影像的可能性.方法 通过基因工程方法,利用大肠杆菌重组表达系统生产并制备PDGFRβ特异性亲和体Z-tri.利用流式细胞术分析Z-tri对体外培养细胞的结合能力,用示踪法监测Z-tri在移植瘤内的细胞分布.将近红外荧光染料CF750与Z-tri偶联制备光学分子探针CF750-Z-tri,并利用光学成像系统检测其对人肺癌细胞移植瘤的显像效果.结果 PDGFRβ特异性亲和体Z-tri在大肠杆菌中获得大量表达,且能通过简单的亲和层析进行纯化.该蛋白体外条件下能与PDGFRβ阳性细胞结合,进入移植瘤内也主要分布于PDGFRβ强阳性细胞.近红外荧光染料CF750能够高效地与Z-tri偶联.制备的近红外荧光探针CF750-Z-tri能快速、清楚地通过光学成像显示肺癌移植瘤.结论 近红外光学分子探针CF750-Z-tri能够用于肺癌分子影像,在利用术中导航指导肺癌组织精准切除方面有应用前景.

The glycoprotein spike (S) on the surface of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites' location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicates that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRβ affibody, which targets platelet-derived growth factor receptor β (PDGFRβ), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRβ fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRβ fused to the IgBD. ZPDGFRβ-modified αPD-L1 bound to both PDGFRβ and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRβ+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRβ fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.