Preclinical development of a novel CCR8/CTLA-4 bispecific antibody for cancer treatment by disrupting CTLA-4 signaling on CD8 T cells and specifically depleting tumor-resident Tregs.

Anti-CTLA-4 antibodies faced challenges due to frequent adverse events and limited efficacy, which spurred the exploration of next-generation CTLA-4 therapeutics to balance regulatory T cells (Tregs) depletion and CD8 T cells activation. CCR8, identified primarily on tumor-infiltrating Tregs, has become a target of interest due to the anti-tumor effects demonstrated by CCR8 antibody-mediated Tregs depletion. Single-cell RNA sequencing analysis reveals that CCR8-positive Tregs constitute a small subset, with concurrent expression of CCR8 and CTLA-4. Consequently, we proposed a novel bispecific antibody targeting CCR8 and CTLA-4 that had the potential to enhance T cell activation while selectively depleting intratumor Tregs. The candidate molecule 2MW4691 was developed in a tetravalent symmetric format, maintaining a strong binding affinity for CCR8 while exhibiting relatively weaker CTLA-4 binding. This selective binding ability allowed 2MW4691 to target and deplete tumor-infiltrating Tregs with higher specificity. In vitro assays verified the antibody's capacity for antibody-dependent cellular cytotoxicity (ADCC) to Tregs with high level of CTLA-4 expression, but not CD8 T cells with relatively low level of CTLA-4 on cell surface. Also, 2MW4691 inhibited the CTLA-4 pathway and enhanced T cell activation. The in vivo therapeutic efficacy of 2MW4691 was further demonstrated using hCCR8 or hCTLA-4 humanized mouse models and hCCR8/hCTLA-4 double knock-in mouse models. In cynomolgus monkeys, 2MW4691 was well-tolerated, exhibited the anticipated pharmacokinetic profile, and had a minimal impact on the peripheral T cell population. The promising preclinical results supported the further evaluation of 2MW4691 as a next-generation Treg-based therapeutics in clinical trials.

LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration.

Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.

Identification of adipocyte plasma membrane-associated protein as a novel modulator of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause disability in newborns and serious clinical diseases in immunocompromised patients. HCMV has a large genome with enormous coding potential; its viral particles are equipped with complicated glycoprotein complexes and can infect a wide range of human cells. Although multiple host cellular receptors interacting with viral glycoproteins have been reported, the mechanism of HCMV infection remains a mystery. Here we report identification of adipocyte plasma membrane-associated protein (APMAP) as a novel modulator active in the early stage of HCMV infection. APMAP is necessary for HCMV infection in both epithelial cells and fibroblasts; knockdown of APMAP expression significantly reduced HCMV infection of these cells. Interestingly, ectopic expression of human APMAP in cells refractory to HCMV infection, such as canine MDCK and murine NIH/3T3 cells, promoted HCMV infection. Furthermore, reduction in viral immediate early (IE) gene transcription at 6 h post infection and delayed nucleus translocation of tegument delivered pp65 at 4 h post infection were detected in APMAP-deficient cells but not in the wildtype cells. These results suggest that APMAP plays a role in the early stage of HCMV infection. Results from biochemical studies of APMAP and HCMV proteins suggest that APMAP could participate in HCMV infection through interaction with gH/gL containing glycoprotein complexes at low pH and mediate nucleus translocation of tegument pp65. Taken together, our results suggest that APMAP functions as a modulator promoting HCMV infection in multiple cell types and is an important player in the complex HCMV infection mechanism.

An HRP-labeled lateral flow immunoassay for rapid simultaneous detection and differentiation of influenza A and B viruses.

Rapid and sensitive diagnosis of influenza is urgently needed to address the limitations of low sensitivity associated with current rapid tests available for clinics and on-site monitoring. A novel horseradish peroxidase (HRP)-labeled lateral flow immunoassay strip (HRP-LFIA) for rapid simultaneous detection and differentiation of influenza A (INF A) and influenza B (INF B) viruses were developed. This immunoassay was based on the signal amplification by the HRP-catalyzed oxidation of 3, 3', 5, 5'-tetramethylbenzidine forming a colored insoluble product, which was proportional to the analyte concentration. Compared with conventional gold-colloidal based strips, an analytical sensitivity enhancement of more than one order of magnitude for thirteen INF virus isolates was observed. A total of 1487 swabs obtained from persons with influenza-like illnesses were tested for the presence of INF A and B viruses using real-time reverse transcription polymerase chain reaction (rRT-PCR) as the reference criterion. The overall sensitivities of HRP-LFIA were 77.5% (100/129) and 71.2% (116/163) for INF A and INF B, respectively. The overall specificities were 99.8% (1144/1146) and 99.8% (918/920), respectively. The nasopharyngeal sampling method yielded higher sensitivity rates of 90.2% (55/61) and 82.6% (71/86). In conclusion, this user-friendly assay could be a promising rapid detection method for rapid screening of INF A and INF B viruses.

Broadband all-light-control with WS2 coated microfibers.

All-optical light amplitude tuning functionality is demonstrated in a layered tungsten disulfide (WS2) nanosheets coated microfiber (MF) structure. Due to the strong light-matter interactions between WS2 nanosheets and the evanescent field around the MF, a large variation in the transmitted power can be observed under both external and internal pump light excitations over a broadband spectrum (~100 nm). A power variation rate of ~0.3744 dB/mW is obtained under external violet pump light excitation, whereas the power variation rate of similar devices in the state of the art are usually less than 0.3 dB/mW. In terms of the response time, a moderate rise/fall time of ∼20.5/19.6 ms is achieved, which is mainly limited by the employed structure fabrication methods. These results indicate that the optical transmitted power of the WS2 coated MF can be modulated by different pump light with the power in the order of mW, thus the proposed device might have potential applications in all optical controllable devices and sensors, etc.

A Novel Anti-LILRB4 CAR-T Cell for the Treatment of Monocytic AML.

To effectively improve treatment for acute myeloid leukemia (AML), new molecular targets and therapeutic approaches need to be identified. Chimeric antigen receptor (CAR)-modified T cells targeting tumor-associated antigens have shown promise in the treatment of some malignancies. However, CAR-T cell development for AML has been limited by lack of an antigen with high specificity for AML cells that is not present on normal hematopoietic stem cells, and thus will not result in myelotoxicity. Here we demonstrate that leukocyte immunoglobulin-like receptor-B4 (LILRB4) is a tumor-associated antigen highly expressed on monocytic AML cells. We generated a novel anti-LILRB4 CAR-T cell that displays high antigen affinity and specificity. These CAR-T cells display efficient effector function in vitro and in vivo against LILRB4+ AML cells. Furthermore, we demonstrate anti-LILRB4 CAR-T cells are not toxic to normal CD34+ umbilical cord blood cells in colony-forming unit assays, nor in a humanized hematopoietic-reconstituted mouse model. Our data demonstrate that anti-LILRB4 CAR-T cells specifically target monocytic AML cells with no toxicity to normal hematopoietic progenitors. This work thus offers a new treatment strategy to improve outcomes for monocytic AML, with the potential for elimination of leukemic disease while minimizing the risk for on-target off-tumor toxicity.

Targeting Human-Cytomegalovirus-Infected Cells by Redirecting T Cells Using an Anti-CD3/Anti-Glycoprotein B Bispecific Antibody.

The host immune response to human cytomegalovirus (HCMV) is effective against HCMV reactivation from latency, though not sufficient to clear the virus. T cells are primarily responsible for the control of viral reactivation. When the host immune system is compromised, as in transplant recipients with immunosuppression, HCMV reactivation and progressive infection can cause serious morbidity and mortality. Adoptive T cell therapy is effective for the control of HCMV infection in transplant recipients. However, it is a highly personalized therapeutic regimen and is difficult to implement in routine clinical practice. In this study, we explored a bispecific-antibody strategy to direct non-HCMV-specific T cells to recognize and exert effector functions against HCMV-infected cells. Using a knobs-into-holes strategy, we constructed a bispecific antibody in which one arm is specific for CD3 and can trigger T cell activation, while the other arm, specific for HCMV glycoprotein B (gB), recognizes and marks HCMV-infected cells based on the expression of viral gB on their surfaces. We showed that this bispecific antibody was able to redirect T cells with specificity for HCMV-infected cells in vitro In the presence of HCMV infection, the engineered antibody was able to activate T cells with no HCMV specificity for cytokine production, proliferation, and the expression of phenotype markers unique to T cell activation. These results suggested the potential of engineered bispecific antibodies, such as the construct described here, as prophylactic or therapeutic agents against HCMV reactivation and infection.

Enzymatic conjugation using branched linkers for constructing homogeneous antibody-drug conjugates with high potency.

Antibody-drug conjugates (ADCs) are emerging therapeutic agents in the treatment of cancer, and various conjugation strategies and chemical linkers have been developed to efficiently construct ADCs. Despite previous extensive efforts for improving conjugation efficiency and ADC homogeneity, most ADC linkers developed to date load only single payloads. Branched linkers that can load multiple payload molecules have yet to be fully explored. It is logical to envisage that a multi-loading strategy allows for increase in drug-to-antibody ratio (DAR) with less chemical or enzymatic modification to the antibody structure compared to traditional linear linkers, leading to efficient ADC construction, minimal destabilization of the antibody structure, and enhanced ADC efficacy. Herein, we report that the branched linkers we designed can be quantitatively installed on an anti-HER2 monoclonal antibody by microbial transglutaminase (MTGase)-mediated conjugation without impairing its antigen binding affinity, enabling modular installation of payload molecules and construction of homogeneous ADCs with increased DARs (up to 8). An anti-HER2 antibody-monomethyl auristatin F conjugate constructed using our branched linkers showed greater in vitro cytotoxicity against HER2-expressing breast cancer cell lines than that consisting of linear linkers, demonstrating the effectiveness of the branched linker-based payload delivery. Our finding demonstrates that enzymatic ADC construction using branched linkers is a promising strategy, which may lead to innovative cancer therapeutics.

Evidence of the Role of R-Spondin 1 and Its Receptor Lgr4 in the Transmission of Mechanical Stimuli to Biological Signals for Bone Formation.

The bone can adjust its mass and architecture to mechanical stimuli via a series of molecular cascades, which have been not yet fully elucidated. Emerging evidence indicated that R-spondins (Rspos), a family of secreted agonists of the Wnt/ß-catenin signaling pathway, had important roles in osteoblastic differentiation and bone formation. However, the role of Rspo proteins in mechanical loading-influenced bone metabolism has never been investigated. In this study, we found that Rspo1 was a mechanosensitive protein for bone formation. Continuous cyclic mechanical stretch (CMS) upregulated the expression of Rspo1 in mouse bone marrow mesenchymal stem cells (BMSCs), while the expression of Rspo1 in BMSCs in vivo was downregulated in the bones of a mechanical unloading mouse model (tail suspension (TS)). On the other hand, Rspo1 could promote osteogenesis of BMSCs under CMS through activating the Wnt/ß-catenin signaling pathway and could rescue the bone loss induced by mechanical unloading in the TS mice. Specifically, our results suggested that Rspo1 and its receptor of leucine-rich repeat containing G-protein-coupled receptor 4 (Lgr4) should be a novel molecular signal in the transmission of mechanical stimuli to biological signal in the bone, and this signal should be in the upstream of Wnt/ß-catenin signaling for bone formation. Rspo1/Lgr4 could be a new potential target for the prevention and treatment of disuse osteoporosis in the future.

Identification of a highly conserved and surface exposed B-cell epitope on the nucleoprotein of influenza A virus.

Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope.

Perforated gastric ulcer causing mediastinal emphysema: A case report.

BACKGROUND: Mediastinal emphysema is a condition in which air enters the mediastinum between the connective tissue spaces within the pleura for a variety of reasons. It can be spontaneous or secondary to chest trauma, esophageal perforation, medically induced factors, etc. Its common symptoms are chest pain, tightness in the chest, and respiratory distress. Most mediastinal emphysema patients have mild symptoms, but severe mediastinal emphysema can cause respiratory and circulatory failure, resulting in serious consequences. CASE SUMMARY: A 75-year-old man, living alone, presented with sudden onset of severe epigastric pain with chest tightness after drinking alcohol. Due to the remoteness of his residence and lack of neighbors, the patient was found by his nephew and brought to the hospital the next morning after the disease onset. Computed tomography (CT) showed free gas in the abdominal cavity, mediastinal emphysema, and subcutaneous pneumothorax. Upper gastrointestinal angiography showed that the esophageal mucosa was intact and the gastric antrum was perforated. Therefore, we chose to perform open gastric perforation repair on the patient under thoracic epidural anesthesia combined with intravenous anesthesia. An operative incision of the muscle layer of the patient's abdominal wall was made, and a large amount of subperitoneal gas was revealed. And a continued incision of the peritoneum revealed the presence of a perforation of approximately 0.5 cm in the gastric antrum, which we repaired after pathological examination. Postoperatively, the patient received high-flow oxygen and cough exercises. Chest CT was performed on the first and sixth postoperative days, and the mediastinal and subcutaneous gas was gradually reduced. CONCLUSION: After gastric perforation, a large amount of free gas in the abdominal cavity can reach the mediastinum through the loose connective tissue at the esophageal hiatus of the diaphragm, and upper gastrointestinal angiography can clarify the site of perforation. In patients with mediastinal emphysema, open surgery avoids the elevation of the diaphragm caused by pneumoperitoneum compared to laparoscopic surgery and avoids increasing the mediastinal pressure. In addition, thoracic epidural anesthesia combined with intravenous anesthesia also avoids pressure on the mediastinum from mechanical ventilation.

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

LILRB3 Supports Immunosuppressive Activity of Myeloid Cells and Tumor Development.

The existing T cell-centered immune checkpoint blockade therapies have been successful in treating some but not all patients with cancer. Immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), that inhibit antitumor immunity and support multiple steps of tumor development are recognized as one of the major obstacles in cancer treatment. Leukocyte Ig-like receptor subfamily B3 (LILRB3), an immune inhibitory receptor containing tyrosine-based inhibitory motifs (ITIM), is expressed solely on myeloid cells. However, it is unknown whether LILRB3 is a critical checkpoint receptor in regulating the activity of immunosuppressive myeloid cells, and whether LILRB3 signaling can be blocked to activate the immune system to treat solid tumors. Here, we report that galectin-4 and galectin-7 induce activation of LILRB3 and that LILRB3 is functionally expressed on immunosuppressive myeloid cells. In some samples from patients with solid cancers, blockade of LILRB3 signaling by an antagonistic antibody inhibited the activity of immunosuppressive myeloid cells. Anti-LILRB3 also impeded tumor development in myeloid-specific LILRB3 transgenic mice through a T cell-dependent manner. LILRB3 blockade may prove to be a novel approach for immunotherapy of solid cancers.

Blockade of dual immune checkpoint inhibitory signals with a CD47/PD-L1 bispecific antibody for cancer treatment.

Background: Even though PD-1/PD-L1 is an identified key "don't find me" signal to active adaptive immune system for cancer treatment, the overall response rate (ORR) for all cancer patients is still limited. Other effective therapeutic modalities to bridge the innate and adaptive immunity to improve ORR are urgently needed. Recently, CD47/SIRPα interaction is confirmed as a critical "don't eat me" signal to active innate immunity. However, the red blood cell (RBC) toxicity is the big concern for the development of CD47-based anti-cancer therapeutics. Methods: Here, we report the development of a CD47/PD-L1 bispecific antibody 6MW3211 to block both PD-1/PD-L1 and CD47/SIRPα signals, and studied the effects of 6MW3211 on anti-tumor immune functions in vitro and in vivo. The pharmacokinetic and toxicity profiles of 6MW3211 were evaluated in GLP non-human primate (NHP) studies. Results: The dual immune checkpoint inhibitory signaling blocker 6MW3211 shows high binding affinity to PD-L1 and low binding affinity to CD47. This inequivalent binding affinity design makes 6MW3211 preferentially bound to PD-L1 on tumor cells followed by disrupting the interaction of CD47/SIRPα. Complex structure determination and flow cytometry assay demonstrated that 6MW3211 has no binding to either human or rhesus monkey RBCs. 6MW3211 effectively blocked both PD-1/DP-L1 and CD47/SIRPα signaling and promoted macrophage phagocytosis of tumor cells. Potent therapeutic efficacies of 6MW3211 in three different mouse models were further observed. Moreover, 6MW3211 was demonstrated to have a fairly good safety profile in a GLP NHP study. In addition, multiplex fluorescent immunohistochemistry (mIHC) staining shows that PD-L1 and CD47 co-express on several different types of human tumor tissues. Conclusions: These results support the development of 6MW3211 for the treatment of PD-L1 and CD47 double positive cancers.

Drynaria Naringin alleviated mechanical stress deficiency-caused bone loss deterioration via Rspo1/Lgr4-mediated Wnt/ß-catenin signalling pathway.

Osteoporosis is a metabolic condition distinguished by the degradation of bone microstructure and mechanical characteristics. Traditional Chinese medicine (TCM) has been employed in China for the treatment of various illnesses. Naringin, an ingredient found in Drynariae TCM, is known to have a significant impact on bone metabolism. For this research, we studied the precise potential effect of Drynaria Naringin on protecting against bone loss caused by stress deficiency. In this study, a tail-suspension (TS) test was performed to establish a mouse model with hind leg bone loss. Some mice received subcutaneous injections of Drynaria Naringin for 30 d. Trabecular bone microarchitecture was evaluated using micro-computed tomography analysis and bone histological analysis. Bone formation and resorption markers were quantified in blood samples from mice or in the supernatant of MC3T3-E1 cells by ELISA analysis, Western blotting, and PCR. Immunofluorescence was utilized to visualize the location of ß-catenin. Additionally, siRNA was employed to knockdown-specific genes in the cells. Our findings highlight the efficacy of Drynaria Naringin in protecting against the deterioration of bone loss and promoting bone formation and Rspo1 expression in a mouse model following the TS test. Specifically, in vitro experiments also indicated that Drynaria Naringin may promote osteogenesis through the Wnt/ß-catenin signalling pathway. Moreover, our results suggest that Drynaria Naringin upregulates the expression of Rspo1/Lgr4, leading to the promotion of osteogenesis via the Wnt/ß-catenin signalling pathway. Therefore, Drynaria Naringin holds potential as a therapeutic medication for osteoporosis. Drynaria Naringin alleviates bone loss deterioration caused by mechanical stress deficiency through the Rspo1/Lgr4-mediated Wnt/ß-catenin signalling pathway.

Oxidative Stress Aggravates Apoptosis of Nucleus Pulposus Cells through m6A Modification of MAT2A Pre-mRNA by METTL16.

The process of intervertebral disc degeneration (IVDD) is complex, and its mechanism is considered multifactorial. Apoptosis of oxidative stressed nucleus pulposus cells (NPCs) should be a fundamental element in the pathogenesis of IVDD. In our pilot study, we found that the expression of MAT2A decreased, and METTL16 increased in the degenerative nucleus pulposus tissues. Previous studies have shown that the balance of splicing, maturation, and degradation of MAT2A pre-mRNA is regulated by METTL16 m6A modification. In the current study, we aimed to figure out whether this mechanism was involved in the aberrant apoptosis of NPCs and IVDD. Human NPCs were isolated and cultured under oxidative stress. An IVDD animal model was established. It showed that significantly higher METTL16 expression and lower MAT2A expression were seen in either the NPCs under oxidative stress or the degenerative discs of the animal model. MAT2A was inhibited with siRNA in vitro or cycloleucine in vivo. METTL16 was overexpressed with lentivirus in vitro or in vivo. Downregulation of MAT2A or upregulation of METTL16 aggravated nucleus pulposus cell apoptosis and disc disorganization. The balance of splicing, maturation, and degradation of MAT2A pre-mRNA was significantly inclined to degradation in the NPCs with the overexpression of METTL16. Increased apoptosis of NPCs under oxidative stress could be rescued by reducing the expression of METTL16 using siRNA with more maturation of MAT2A pre-mRNA. Collectively, oxidative stress aggravates apoptosis of NPCs through disrupting the balance of splicing, maturation, and degradation of MAT2A pre-mRNA, which is m6A modified by METTL16.

Blocking LAIR1 signaling in immune cells inhibits tumor development.

The current immune checkpoint blockade therapy has been successful in treating some cancers but not others. New molecular targets and therapeutic approaches of cancer immunology need to be identified. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) is an immune inhibitory receptor expressing on most immune cell types. However, it remains a question whether we can specifically and actively block LAIR1 signaling to activate immune responses for cancer treatment. Here we report the development of specific antagonistic anti-LAIR1 monoclonal antibodies and studied the effects of LAIR1 blockade on the anti-tumor immune functions. The anti-LAIR1 antagonistic antibody stimulated the activities of T cells, natural killer cells, macrophages, and dendritic cells in vitro. The single-cell RNA sequencing analysis of intratumoral immune cells in syngeneic human LAIR1 transgenic mice treated with control or anti-LAIR1 antagonist antibodies indicates that LAIR1 signaling blockade increased the numbers of CD4 memory T cells and inflammatory macrophages, but decreased those of pro-tumor macrophages, regulatory T cells, and plasmacytoid dendritic cells. Importantly, the LAIR1 blockade by the antagonistic antibody inhibited the activity of immunosuppressive myeloid cells and reactivated T cells from cancer patients in vitro and impeded tumor metastasis in a humanized mouse model. Blocking LAIR1 signaling in immune cells represents a promising strategy for development of anti-cancer immunotherapy.

Pre-Clinical Development of a Potent Neutralizing Antibody MW3321 With Extensive SARS-CoV-2 Variants Coverage.

Since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, several variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and have consistently replaced the previous dominant variant. Therapeutics against variants of SARS-CoV-2 are urgently needed. Ideal SARS-CoV-2 therapeutic antibodies would have high potency in viral neutralization against several emerging variants. Neutralization antibodies targeting SARS-CoV-2 could provide immediate protection after SARS-CoV-2 infection, especially for the most vulnerable populations. In this work, we comprehensively characterize the breadth and efficacy of SARS-CoV-2 RBD-targeting fully human monoclonal antibody (mAb) MW3321. MW3321 retains full neutralization activity to all tested 12 variants that have arisen in the human population, which are assigned as VOC (Variants of Concern) and VOI (Variants of Interest) due to their impacts on public health. Escape mutation experiments using replicating SARS-CoV-2 pseudovirus show that escape mutants were not generated until passage 6 for MW3321, which is much more resistant to escape mutation compared with another clinical staged SARS-CoV-2 neutralizing mAb MW3311. MW3321 could effectively reduce viral burden in hACE2-transgenic mice challenged with either wild-type or Delta SARS-CoV-2 strains through viral neutralization and Fc-mediated effector functions. Moreover, MW3321 exhibits a typical hIgG1 pharmacokinetic and safety profile in cynomolgus monkeys. These data support the development of MW3321 as a monotherapy or cocktail against SARS-CoV-2-related diseases.

Antibody-dependent enhancement (ADE) of SARS-CoV-2 pseudoviral infection requires FcγRIIB and virus-antibody complex with bivalent interaction.

Understanding the underlying molecular mechanisms behind ADE of SARS-CoV-2 is critical for development of safe and effective therapies. Here, we report that two neutralizing mAbs, MW01 and MW05, could enhance the infection of SARS-CoV-2 pseudovirus on FcγRIIB-expressing B cells. X-ray crystal structure determination and S trimer-binding modeling showed that MW01 and MW05 could bind to RBDs in S trimer with both "up" and "down" states. While, the neutralizing mAb MW07, which has no ADE activity only binds to RBD in S trimer with "up" state. Monovalent MW01 and MW05 completely diminished the ADE activity compared with their bivalent counterparts. Moreover, both macropinocytosis and endocytosis are confirmed involving in ADE of SARS-CoV-2 pseudoviral infection. Blocking endosome transportation and lysosome acidification could inhibit the ADE activity mediated by MW05. Together, our results identified a novel ADE mechanism of SARS-CoV-2 pseudovirus in vitro, FcγRIIB-mediated uptake of SARS-CoV-2/mAb complex with bivalent interaction.

Characterization of MW06, a human monoclonal antibody with cross-neutralization activity against both SARS-CoV-2 and SARS-CoV.

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.