Multienzyme-Like Nanozyme Encapsulated Ocular Microneedles for Keratitis Treatment.

Keratitis, an inflammation of the cornea caused by bacterial or fungal infections, is one of the leading causes of severe visual disability and blindness. Keratitis treatment requires both the prevention of infection and the reduction of inflammation. However, owing to their limited therapeutic functions, in addition to the ocular barrier, existing conventional medications are characterized by poor efficacy and low bioavailability, requiring high dosages or frequent topical treatment, which represents a burden on patients and increases the risk of side effects. In this study, manganese oxide nanocluster-decorated graphdiyne nanosheets (MnOx/GDY) are developed as multienzyme-like nanozymes for the treatment of infectious keratitis and loaded into hyaluronic acid and polymethyl methacrylate-based ocular microneedles (MGMN). MGMN not only exhibits antimicrobial and anti-inflammatory effects owing to its multienzyme-like activities, including oxidase, peroxidase, catalase, and superoxide dismutase mimics but also crosses the ocular barrier and shows increased bioavailability via the microneedle system. Moreover, MGMN is demonstrated to eliminate pathogens, prevent biofilm formation, reduce inflammation, alleviate ocular hypoxia, and promote the repair of corneal epithelial damage in in vitro, ex vivo, and in vivo experiments, thus providing a better therapeutic effect than commercial ophthalmic voriconazole, with no obvious microbial resistance or cytotoxicity.

Interaction of species A rotavirus VP4 with the cellular proteins vimentin and actin related protein 2 discovered by a proximity interactome assay.

IMPORTANCE: Rotavirus (RV) is an important zoonosis virus, which can cause severe diarrhea and extra-intestinal infection. To date, some proteins or carbohydrates have been shown to participate in the attachment or internalization of RV, including HGBAs, Hsc70, and integrins. This study attempted to indicate whether there were other proteins that would participate in the entry of RV; thus, the RV VP4-interacting proteins were identified by proximity labeling. After analysis and verification, it was found that VIM and ACTR2 could significantly promote the proliferation of RV in intestinal cells. Through further viral binding assays after knockdown, antibody blocking, and recombinant protein overexpression, it was revealed that both VIM and ACTR2 could promote RV replication.

The learning curve for modified hand-assisted retroperitoneoscopic living donor nephrectomy.

BACKGROUND: We aimed to introduce our modified hand-assisted retroperitoneoscopic living donor nephrectomy (HARPLDN) technique and define the learning curve. METHODS: One hundred thirty-eight kidney donors who underwent modified HARPLDN by the same surgeon between May 2015 and March 2022 were included. A cumulative sum (CUSUM) learning curve analysis was performed with the total operation time as the study outcome. RESULTS: In total, the mean operative time was 138.2 ± 32.1 min. The median warm ischemic time (WIT) and estimated blood loss were 90 s and 50 ml, respectively. The learning curve for the total operative time was best modeled as a second-order polynomial with the following equation: CUSUMOT (min) = (-0.09 case number2) + (12.88 case number) - 67.77 (R2 = 0.7875; p<0.05). The CUSUM learning curve included the following three unique phases: phase 1 (the initial 41 cases), representing the initial learning curve; phase 2 (the middle 43 cases), representing expert competence; and phase 3 (the final 54 cases), representing mastery. The overall 6-month graft survival rate was 99.3%, with 94.9% immediate onset of graft function without delayed graft function and 0.7% ureteral complications. CONCLUSIONS: Our modified method is safe and effective for living donor nephrectomy and has the advantages of a shorter operating time and optimized WIT. The surgeon can become familiar with the modified HARPLDN after 41 cases and effectively perform the next 97 cases.

Interaction of Nipah Virus F and G with the Cellular Protein Cortactin Discovered by a Proximity Interactome Assay.

Nipah virus (NiV) is a highly lethal zoonotic virus with a potential large-scale outbreak, which poses a great threat to world health and security. In order to explore more potential factors associated with NiV, a proximity labeling method was applied to investigate the F, G, and host protein interactions systematically. We screened 1996 and 1524 high-confidence host proteins that interacted with the NiV fusion (F) glycoprotein and attachment (G) glycoprotein in HEK293T cells by proximity labeling technology, and 863 of them interacted with both F and G. The results of GO and KEGG enrichment analysis showed that most of these host proteins were involved in cellular processes, molecular binding, endocytosis, tight junction, and other functions. Cytoscape software (v3.9.1) was used for visual analysis, and the results showed that Cortactin (CTTN), Serpine mRNA binding protein 1 (SERBP1), and stathmin 1 (STMN1) were the top 20 proteins and interacted with F and G, and were selected for further validation. We observed colocalization of F-CTTN, F-SERBP1, F-STMN1, G-CTTN, G-SERBP1, and G-STMN1 using confocal fluorescence microscopy, and the results showed that CTTN, SERBP1, and STMN1 overlapped with NiV F and NiV G in HEK293T cells. Further studies found that CTTN can significantly inhibit the infection of the Nipah pseudovirus (NiVpv) into host cells, while SERBP1 and STMN1 had no significant effect on pseudovirus infection. In addition, CTTN can also inhibit the infection of the Hendra pseudovirus (HeVpv) in 293T cells. In summary, this study revealed that the potential host proteins interacted with NiV F and G and demonstrated that CTTN could inhibit NiVpv and HeVpv infection, providing new evidence and targets for the study of drugs against these diseases.

Origin of the lamina propria dendritic cell network.

CX(3)CR1(+) and CD103(+) dendritic cells (DCs) in intestinal lamina propria play a key role in mucosal immunity. However, the origin and the developmental pathways that regulate their differentiation in the lamina propria remain unclear. We showed that monocytes gave rise exclusively to CD103(-)CX(3)CR1(+) lamina propria DCs under the control of macrophage-colony-stimulating factor receptor (M-CSFR) and Fms-like thyrosine kinase 3 (Flt3) ligands. In contrast, common DC progenitors (CDP) and pre-DCs, which give rise to lymphoid organ DCs but not to monocytes, differentiated exclusively into CD103(+)CX(3)CR1(-) lamina propria DCs under the control of Flt3 and granulocyte-macrophage-colony-stimulating factor receptor (GM-CSFR) ligands. CD103(+)CX(3)CR1(-) DCs but not CD103(-)CX(3)CR1(+) DCs in the lamina propria constitutively expressed CCR7 and were the first DCs to transport pathogenic Salmonella from the intestinal tract to the mesenteric lymph nodes. Altogether, these results underline the diverse origin of the lamina propria DC network and identify mucosal DCs that arise from pre-DCs as key sentinels of the gut immune system.

Spatiotemporal expression of endogenous TLR4 ligands leads to inflammation and bone erosion in mouse collagen-induced arthritis.

Increased expression of endogenous Toll-like receptor 4 (TLR4) ligands (e.g., Tenascin-C, S100A8/A9, citrullinated fibrinogen (cFb) immune complexes) has been observed in patients with rheumatoid arthritis (RA). However, their roles in RA pathogenesis are not well understood. Here, we investigated the expression kinetics and role of endogenous TLR4 ligands in the murine model of collagen-induced arthritis (CIA). Tenascin-C was upregulated in blood early in CIA, and correlated positively with the clinical score at day 56. Levels of S100A8/A9 increased starting from day 28, peaking at day 42, and correlated positively with joint inflammation. Levels of anti-cFb antibodies increased during the late phase of CIA and correlated positively with both joint inflammation and cartilage damage. Blockade of TLR4 activation at the time of the first TLR4 ligand upregulation prevented clinical and histological signs of arthritis. A TLR4-dependent role was also observed for Tenascin-C and cFb immune complexes in osteoclast differentiation in vitro. Taken together, our data suggests that the pathogenic contribution of TLR4 in promoting joint inflammation and bone erosion during CIA occurs via various TLR4 ligands arising at different stages of disease. The data also suggests that Blockade of TLR4 with monoclonal antibodies is a promising strategy in RA treatment.

Interactome map reveals phospholipid scramblase 1 as a novel regulator of hepatitis B virus x protein.

HBV X protein plays crucial roles during viral infection and hepatocellular carcinoma (HCC) development through interaction with various host factors. Here, we mapped the interactome of HBx using a yeast two-hybrid screen. Nine human proteins were identified as novel interacting partners of HBx, one of which is phospholipid scramblase 1 (PLSCR1). PLSCR1 is an interferon-inducible protein that mediates antiviral activity against DNA and RNA viruses. However, the molecular mechanisms of PLSCR1 activity against HBV remain unclear. Here, we reported that PLSCR1 promotes HBx degradation by a proteasome- and ubiquitin-dependent mechanism. Furthermore, we found that PLSCR1 inhibits HBx-mediated cell proliferation. After HBV infection, the protein level of PLSCR1 in plasma is elevated, and chronic hepatitis B patients with low plasma levels of PLSCR1 have a high risk of developing HCC. These results suggest that the nuclear trafficking of PLSCR1 mediates the antiviral activity and anticarcinogenesis against HBV by regulating HBx stability.

Selective antibody intervention of Toll-like receptor 4 activation through Fc γ receptor tethering.

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

Posterior short segment pedicle screw fixation and TLIF for the treatment of unstable thoracolumbar/lumbar fracture.

BACKGROUND: Currently, Posterior Short Segment Pedicle Screw Fixation is a popular procedure for treating unstable thoracolumbar/lumbar burst fracture. But progressive kyphosis and a high rate of hardware failure because of lack of the anterior column support remains a concern. The efficacy of different methods remains debatable and each technique has its advantages and disadvantages. METHODS: A consecutive series of 20 patients with isolated thoracolumbar/lumbar burst fractures were treated by posterior short segment pedicle screw fixation and transforaminal thoracolumbar/lumbar interbody fusion (TLIF) between January 2005 and December 2007. All patients were followed up for a minimum of 2 years. Demographic data, neurologic status, anterior vertebral body heights, segmental Cobb angle and treatment-related complications were evaluated. RESULTS: The mean operative time was 167 minutes (range, 150-220). Blood loss was 450 ~ 1200 ml, an average of 820 ml. All patients recovered with solid fusion of the intervertebral bone graft, without main complications like misplacement of the pedicle screw, nerve or vessel lesion or hard ware failure. The post-operative radiographs demonstrated a good fracture reduction and it was well maintained until the bone graft fusion. Neurological recovery of one to three Frankel grade was seen in 14 patients with partial neurological deficit, three grades of improvement was seen in one patient, two grades of improvement was observed in 6 patients and one grade of improvement was found in 6 patients. All the 6 patients with no paraplegia on admission remained neurological intact, and in one patient with Frankel D on admission no improvement was observed. CONCLUSION: Posterior short-segment pedicle fixation in conjunction with TLIF seems to be a feasible option in the management of selected thoracolumbar/lumbar burst fractures, thereby addressing all the three columns through a single approach with less trauma and good results.

Ultrasound-Augmented Multienzyme-like Nanozyme Hydrogel Spray for Promoting Diabetic Wound Healing.

Treatment of diabetic foot ulcers (DFU) needs to reduce inflammation, relieve hypoxia, lower blood glucose, promote angiogenesis, and eliminate pathogenic bacteria, but the therapeutic efficacy is greatly limited by the diversity and synergy of drug functions as well as the DFU microenvironment itself. Herein, an ultrasound-augmented multienzyme-like nanozyme hydrogel spray was developed using hyaluronic acid encapsulated l-arginine and ultrasmall gold nanoparticles and Cu1.6O nanoparticles coloaded phosphorus doped graphitic carbon nitride nanosheets (ACPCAH). This nanozyme hydrogel spray possesses five types of enzyme-like activities, including superoxide dismutase (SOD)-, catalase (CAT)-, glucose oxidase (GOx)-, peroxidase (POD)-, and nitric oxide synthase (NOS)-like activities. The kinetics and reaction mechanism of the sonodynamic/sonothermal synergistic enhancement of the SOD-CAT-GOx-POD/NOS cascade reaction of ACPCAH are fully investigated. Both in vitro and in vivo tests demonstrate that this nanozyme hydrogel spray can be activated by the DFU microenvironment to reduce inflammation, relieve hypoxia, lower blood glucose, promote angiogenesis, and eliminate pathogenic bacteria, thus accelerating diabetic wound healing effectively. This study highlights a competitive approach based on multienzyme-like nanozymes for the development of all-in-one DFU therapies.

Ultrasound activatable microneedles for bilaterally augmented sono-chemodynamic and sonothermal antibacterial therapy.

Chemodynamic therapy (CDT) employs Fenton catalysts to kill bacteria by converting hydrogen peroxide (H2O2) into toxic hydroxyl radical (•OH). Among them, Fenton-type metal peroxide nanoparticles fascinate nanomaterials with intriguing physiochemical properties, but research on this antibacterial agent is still in its infancy. Herein, a distinct CuO2/TiO2 heterostructure constituted of ultrasmall copper peroxide (CuO2) nanoclusters and sonosensitized ultrathin oxygen vacancy-rich porous titanium oxide (OV-TiO2) nanosheets was developed and was incorporated into microneedles for bilaterally augmented sono-chemodynamic and sonothermal antibacterial therapy. Engineering CuO2 nanoclusters on the surface of TiO2 nanosheets not only endows the Fenton catalytic activity for sono-chemodynamic therapy (SCDT), but also improves the sonodynamic and sonothermal performance of TiO2 by narrowing the bandgap of TiO2 and suppressing the recombination of electron-hole pairs. The high efficacy of this CuO2/TiO2 integrated microneedle (CTMN) patch was systematically demonstrated both in vitro and in vivo with the eliminating rate >99.9999% against multidrug resistant (MDR) pathogens in 5 min as well as accelerated wound tissue healing. This work highlights a promisingly new and efficient strategy for the development of sonosensitive and chemoreactive nanomedicine for non-antibiotic therapies. STATEMENT OF SIGNIFICANCE: Feton-type metal peroxides, a novel nanomaterial with self-supplied oxygen and hydrogen peroxide, can achieve effective antimicrobial activity in vitro. However, there is a lack of effective nanomaterial delivery systems and suitable means for in vivo activation/enhancement of antimicrobial activity during bacterial infected skin wound treatment. In this study, we designed and prepared efficient ultrasound activable microneedles that effectively addressed the deficiencies mentioned above and established a new paradigm for efficient utilization of metal peroxide nanomaterials and ultrasound based strategies. Noticeably, copper peroxide nanoclusters/oxygen vacancy-rich porous titanium oxide nanosheets (CuO2/TiO2) integrated microneedle (CTMN) patch combines advantages of both sono-chemodynamic and sonothermal antibacterial therapy, achieving one of the most instant and effective antibacterial efficacy (>99.9999% in 5 min) in vivo reported till now.

Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers.

BACKGROUND: T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). METHODS: We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). RESULTS: NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. CONCLUSIONS: In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.

Although IL-6 trans-signaling is sufficient to drive local immune responses, classical IL-6 signaling is obligate for the induction of T cell-mediated autoimmunity.

IL-6-mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6-mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Detection of Leishmania donovani infection using magnetic beads-based serum peptide profiling by MALDI-TOF MS in mice model.

Leishmaniasis is an important parasitic disease, and definite diagnosis using a specific and sensitive method is the first step to cure the disease. Here, we present a novel diagnostic strategy based on serum peptide profiling by magnetic beads and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The serum peptides from the Leishmani donovani-infected and healthy mice were enriched by the optimized magnetic beads. The mass spectrograms were acquired by MALDI-TOF MS and analyzed by the ClinProTools bioinformatics software from Bruker Daltonics. The diagnostic model of serum peptide profiling produced by the ClinProTools software could correctly detect L. donovani infection in mice from the third day post-infection, with the accuracy of 94.1%, sensitivity of 92.4%, and specificity of 97.1%, respectively. The results of the present study suggested that the serum peptide profiling by MALDI-TOF MS is a novel potential tool for the clinical diagnosis of leishmaniasis.

Effect of Carbapenem-Resistant Klebsiella pneumoniae Infection on the Clinical Outcomes of Kidney Transplant Recipients.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection has proven to be difficult to control and typically presents with devastating effects. Methods: This retrospective study was conducted on the renal recipients at our institution between January 2021 to January 2022. Clinical data was collected to identify factors associated with CRKP infection and clinical outcomes. Results: There were 104 cases out of 186 total renal recipients who presented with at least one infection within 3 months after KT, and 14 cases developed unfavorable clinical outcomes. We identified 16 confirmed CRKP infected cases with the incidence of 8.60%. Possible donor derived infection (DDI) (OR = 6.743; 95% CI: 1.477-30.786; P = 0.014) were independent risk factors for the occurrence of CRKP infection of renal recipients in our analysis, CRKP infection (OR = 20.723; 95% CI: 3.448-124.547; P = 0.001) and pneumonia (OR = 28.458; 95% CI: 1.956-413.984 P = 0.014) were independent risk factors for the occurrence of unfavorable clinical outcomes following KT, and the occurrence of unfavorable clinical outcomes following KT were significantly associated with CRKP infection (r = 0.535; P < 0.001) and antibiotic regimen containing ceftazidime/avibactam (CZA) (r = -0.655; P = 0.006). The use of CZA was significantly different in the comparison of antibiotic regimens between the CRKP infected renal recipients with unfavorable outcomes and CRKP infected patients with favorable outcomes. Conclusion: It is possible that DDI can lead to CRKP infection, and CRKP infection and pneumonia were closely correlated with poor prognosis. The use of CZA may play a role in avoiding the unfavorable outcomes of CRKP infected recipients.

Corynebacterium striatum Endocarditis After Renal Transplantation Confirmed by Metagenomic Next-Generation Sequencing: Case Report and Literature Review.

Background: Potent immune-suppressive therapy has been demonstrated to increase the risk of infective endocarditis (IE) in renal recipients. Reports of Corynebacterium striatum (C. striatum) endocarditis in renal recipients are scarce, thus limiting understanding of the disease. Case Presentation: We describe a case of native valve endocarditis caused by C. striatum in a 35-year-old male patient. The young man with end-stage renal failure underwent kidney transplantation because of autosomal dominant polycystic kidney disease. Ceftazidime was administered after the surgery according to routine procedures, and the patient was discharged on the 14th day after the surgery without any evidence of infection. The patient experienced fever on the 56th day, and Corynebacterium was cultured from the patient's blood, in agreement with the results of testing of the donor kidney preservation solution. On the 64th day, multiple thromboses were found in the right external iliac artery, particularly around the anastomotic orifice of the transplanted renal artery. Vegetation was found in the posterior mitral valve tip on the 65th day. The patient had symptoms of persistent angina pectoris and chest tightness and underwent mitral valve replacement and vegetative resection. The patient eventually died. C. striatum was detected in the mitral valve and vegetation tissue with metagenomic next-generation sequencing. Conclusion: C. striatum may cause endocarditis and endanger patients' lives and thus warrants greater attention. Genotypic assays such as metagenomic next-generation sequencing are demonstrated to be effective in confirming species identity. Adequate anti-infection therapy and early surgery are required after IE is discovered.

An antibody-based proximity labeling protocol to identify biotinylated interactors of SARS-CoV-2.

Elucidating the molecular interactions between virus and host is fundamental to understanding the mechanism of viral pathogenesis. Here, we present a protocol to screen SARS-CoV-2 protein interactors using an antibody-based TurboID proximity labeling approach. This technique directly identifies biotinylated peptides labeled by the TurboID-tagged viral proteins. We describe the steps to prepare biotinylated peptide samples for mass spectrometry analysis and a stringent workflow to identify biotinylated high-confidence interactors of the virus by filtering out non-specific co-purified proteins. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping.

Genome-wide physical protein-protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.

CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model.

BACKGROUND: CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS: The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments. RESULTS: We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS: The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).