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Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/β-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

The prosperity of the biotherapeutics market reflects the feasibility and effectiveness of therapeutic antibodies for the treatment of cancers, inflammatory disorders, and refractory infections. As drawbacks emerge in clinical trials and practice, such as impeded binding, reduced effector functions, and frequent adverse reactions, modifications of therapeutic antibodies are unprecedently burgeoning in research and development (R&D). These modifications include: ① modified glycosylation; ② fragment of crystallizable domain (Fc) amino acid alterations; ③ cross-isotype or cross-subclass exchanges; ④ antibody–drug conjugates (ADCs); ⑤ single chain of variable region fragment (scFv) for chimeric antigen receptor T (CAR-T) cells; and ⑥ bispecific antibodies (bsAbs) in order to promote binding affinity, half-life in circulation, effectiveness toward target cells and, ultimately, to achieve overall improved efficacy. While many achievements have been made around the world in the past decades, China has been playing an active role in this realm, with its great demand for biotherapeutics with R&D potential. This review recapitulates the international progress that has been achieved with modified therapeutic antibodies, and then focuses on that of China in an independent section.

Abstract Major gaps in understanding the molecular mechanisms of colorectal cancer (CRC) progression and intestinal mucosal repair have hampered therapeutic development for gastrointestinal disorders. Trefoil factor 3 (TFF3) has been reported to be involved in CRC progression and intestinal mucosal repair; however, how TFF3 drives tumors to become more aggressive or metastatic and how TFF3 promotes intestinal mucosal repair are still poorly understood. Here, we found that the upregulated TFF3 in CRC predicted a worse overall survival rate. TFF3 deficiency impaired mucosal restitution and adenocarcinogenesis. CD147, a membrane protein, was identified as a binding partner for TFF3. Via binding to CD147, TFF3 enhanced CD147-CD44s interaction, resulting in signal transducer and activator of transcription 3 (STAT3) activation and prostaglandin G/H synthase 2 (PTGS2) expression, which were indispensable for TFF3-induced migration, proliferation, and invasion. PTGS2-derived PGE2 bound to prostaglandin E2 receptor EP4 subtype (PTGER4) and contributed to TFF3-stimulated CRC progression. Solution NMR studies of the TFF3-CD147 interaction revealed the key residues critical for TFF3 binding and the induction of PTGS2 expression. The ability of TFF3 to enhance mucosal restitution was weakened by a PTGS2 inhibitor. Blockade of TFF3-CD147 signaling using competitive inhibitory antibodies or a PTGS2 inhibitor reduced CRC lung metastasis in mice. Our findings bring strong evidence that CD147 is a novel receptor for TFF3 and PTGS2 signaling is critical for TFF3-induced mucosal restitution and CRC progression, which widens and deepens the understanding of the molecular function of trefoil factors.

Abstract Opioid abuse and addiction have become a global pandemic, posing tremendous health and social burdens. The rewarding effects and the occurrence of withdrawal symptoms are the two mainstays of opioid addiction. Mu‐opioid receptors (MORs), a member of opioid receptors, play important roles in opioid addiction, mediating both the rewarding effects of opioids and opioid withdrawal syndrome (OWS). The underlying mechanism of MOR‐mediated opioid rewarding effects and withdrawal syndrome is of vital importance to understand the nature of opioid addiction and also provides theoretical basis for targeting MORs to treat drug addiction. In this review, we first briefly introduce the basic concepts of MORs, including their structure, distribution in the nervous system, endogenous ligands, and functional characteristics. We focused on the brain circuitry and molecular mechanism of MORs‐mediated opioid reward and withdrawal. The neuroanatomical and functional elements of the neural circuitry of the reward system underlying opioid addiction were thoroughly discussed, and the roles of MOR within the reward circuitry were also elaborated. Furthermore, we interrogated the roles of MORs in OWS, along with the structural basis and molecular adaptions of MORs‐mediated withdrawal syndrome. Finally, current treatment strategies for opioid addiction targeting MORs were also presented.

Abstracts Recent evidence suggests that CD147 serves as a novel receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Blocking CD147 via anti-CD147 antibody could suppress the in vitro SARS-CoV-2 replication. Meplazumab is a humanized anti-CD147 IgG2 monoclonal antibody, which may effectively prevent SARS-CoV-2 infection in coronavirus disease 2019 (COVID-19) patients. Here, we conducted a randomized, double-blinded, placebo-controlled phase 1 trial to evaluate the safety, tolerability, and pharmacokinetics of meplazumab in healthy subjects, and an open-labeled, concurrent controlled add-on exploratory phase 2 study to determine the efficacy in COVID-19 patients. In phase 1 study, 59 subjects were enrolled and assigned to eight cohorts, and no serious treatment-emergent adverse event (TEAE) or TEAE grade ≥3 was observed. The serum and peripheral blood C max and area under the curve showed non-linear pharmacokinetic characteristics. No obvious relation between the incidence or titer of positive anti-drug antibody and dosage was observed in each cohort. The biodistribution study indicated that meplazumab reached lung tissue and maintained >14 days stable with the lung tissue/cardiac blood–pool ratio ranging from 0.41 to 0.32. In the exploratory phase 2 study, 17 COVID-19 patients were enrolled, and 11 hospitalized patients were involved as concurrent control. The meplazumab treatment significantly improved the discharged (P = 0.005) and case severity (P = 0.021), and reduced the time to virus negative (P = 0.045) in comparison to the control group. These results show a sound safety and tolerance of meplazumab in healthy volunteers and suggest that meplazumab could accelerate the recovery of patients from COVID-19 pneumonia with a favorable safety profile.

Abstract Systemic inflammatory response syndrome (SIRS) is characterized by dysregulated cytokine release, immune responses and is associated with organ dysfunction. IL‐6R blockade indicates promising therapeutic effects in cytokine release storm but still remains unknown in SIRS. To address the issue, we generated the human il‐6r knock‐in mice and a defined epitope murine anti‐human membrane‐bound IL‐6R (mIL‐6R) mAb named h‐mIL‐6R mAb. We found that the h‐mIL‐6R and the commercial IL‐6R mAb Tocilizumab significantly improved the survival rate, reduced the levels of TNF‐α, IL‐6, IL‐1β, IFN‐γ, transaminases and blood urea nitrogen of LPS‐induced SIRS mice. Besides, the h‐mIL‐6R mAb could also dramatically reduce the levels of inflammatory cytokines in LPS‐treated THP‐1 cells in vitro. RNA‐seq analysis indicated that the h‐mIL‐6R mAb could regulate LPS‐induced activation of NF‐κB/Ccl2 and NOD‐like receptor signaling pathways. Furthermore, we found that the h‐mIL‐6R mAb could forwardly inhibit Ccl2 expression and NLRP3‐mediated pyroptosis by suppressing NF‐κB in combination with the NF‐κB inhibitor. Collectively, mIL‐6R mAbs suppressed NF‐κB/Ccl2 signaling and inflammasome activation. IL‐6R mAbs are potential alternative therapeutics for suppressing excessive cytokine release, over‐activated inflammatory responses and alleviating organ injuries in SIRS.

Recently, immunotherapy targeting tumor-infiltrating lymphocytes (TILs) has emerged as a critical and promising treatment in several types of cancer. However, not all cancer types have been tested in immunotherapeutic trials, and different patients and cancer types may have unpredictable clinical outcomes. This situation has created a particular exigency for analyzing the prognostic significance of tumor-infiltrating T cells (TIL-T) and B cells (TIL-B) across different cancer types. To address the critical role of TILs, the abundances of TIL-T and TIL-B cells, as determined by the protein levels of LCK and CD20, were analyzed across heterogeneous human malignancies. TIL-T and TIL-B cells showed varying prognostic significances across heterogeneous cancer types. Additionally, distinct distributions of TIL-T and TIL-B cells were observed in different cancer and tumor microenvironment (TME) subtypes. Next, we analyzed the cellular context for the TME communication network involving the well-acknowledgeable chemokine receptors of TIL-T and TIL-B cells, implying the functional interactions with TME. Additionally, these chemokine receptors, expressed by TIL-T and TIL-B cells, were remarkably correlated with the levels of TIL-T or TIL-B cell infiltrations across nearly all the cancer types, indicating these chemokine receptors as universal targets for up- and down-regulating the TIL-T and TIL-B cells. Lastly, we provide the prognostic landscape of TIL-T and TIL-B cells across 30 cancer types and the subgroups defined by gender, histopathology, histological grade, therapeutic approach, drug, and TME subtype, which are intended to be a resource to fuel the investigations of TILs, with important implications for cancer immunotherapy.

CD147 is highly expressed on the surface of numerous tumor cells to promote invasion and metastasis. Targeting these cells with CD147-specific antibodies has been validated as an effective approach for lung and liver cancer therapy. In the immune system, CD147 is recognized as a co-stimulatory receptor and impacts the outcome of thymic selection. Using T cell-specific deletion, we showed here that in thymus CD147 is indispensable for the stable αβ T cell lineage commitment: loss of CD147 biases both multipotent DN (double negative) and fully committed DP (double positive) cells into innate NK-like lineages. Mechanistically, CD147 deficiency results in impaired Wnt signaling and expression of BCL11b, a master transcription factor in determining T cell identity. In addition, functional blocking of CD147 by antibody phenocopies genetic deletion to enrich NK-like cells in the periphery. Furthermore, using a melanoma model and orthotopic liver cancer transplants, we showed that the augmentation of NK-like cells strongly associates with resistance against tumor growth upon CD147 suppression. Therefore, besides its original function in tumorigenesis, CD147 is also an effective surface target for immune modulation in tumor therapy.

Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer-related death in the Asia-Pacific region. Our previous work showed that knockdown of CD98 significantly inhibits malignant HCC cell phenotypes in vitro and in vivo. The level of CD98 in the membrane is tightly regulated to mediate complex processes associated with cell-cell communication and intracellular signaling. In addition, the intracellular domain of CD98 (CD98-ICD) seems to be of vital importance for recycling CD98 to the membrane after it is endocytosed. The intracellular and transmembrane domains of CD98 associate with β-integrins (primarily β1 but also β3), and this association is essential for CD98 mediation of integrin-like signaling and complements dominant suppression of β1-integrin. We speculated that isolated CD98-ICD would similarly suppress β1-integrin activation and inhibit the malignant behaviors of cancer cells. In particular, the exact role of CD98-ICD has not been studied independently in HCC. In this study, we found that ectopic expression of CD98-ICD inhibited the malignant phenotypes of HCC cells, and the mechanism possibly involves β1-integrin suppression. Moreover, the expression levels of CD98, β1-integrin-A (the activated form of β1-integrin) and Ki-67 were significantly increased in HCC tissues relative to those of normal liver tissues. Therefore, our preliminary study indicates that ectopic CD98-ICD has an inhibitory role in the malignant development of HCC, and shows that CD98-ICD acts as a dominant negative mutant of CD98 that attenuates β1-integrin activation. CD98-ICD may emerge as a promising candidate for antitumor treatment. [ABSTRACT FROM AUTHOR]

Background: Dysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer. Basigin can enhance cancer progression, but its precise mechanism remains unclear. CD98 promotes cell spreading and tumorigenicity by triggering integrin clustering and enhancing cell adhesion to the extracellular matrix. The endocytosis and recyle of basigin and CD98 might play critical roles in cancer. Methods: The role of CD98 was confirmed in liver cancer cells by cell spreading in vitro and tumorigenicity by nude mice xenograft tumor assay in vivo; membrane expression of basigin and CD98 in SMMC-7721 was measured by FCAS; pull down and SPR analysis were uses to reveal the direct association between basigin and CD98; DsRed1 tagged CD98 was blocked in the cytoplasm in K7721 (whose basigin was knockn out) and had a well colocalization with ER and Rab5a positive recycling endosomes under co-focal; finally, by FRET imaging and FCAS we observed the internalization of basigin and CD98 was flotillin-1-regulated, and their recycle at early steps was Arf6-mediated. Results: Basigin and CD98 were highly expressed and co-localized on the human hepatocellular carcinoma (HCC) cell membrane; basigin can directly bind to CD98, mediating CD98 redistribution on the HCC cell membrane and activating the downstream integrin signaling pathway. Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6. This recycling process for basigin and CD98 promotes cell spreading and tumor growth in liver cancer xenografts. Conclusion: Basigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Aims: To evaluate HAb18G/CD147 as a cancer-associated biomarker using its monoclonal antibody HAb18. Methods and results: On immunohistochemical analysis of 28 tissue microarrays and pathological sections of 1117 breast tissue samples, HAb18G/CD147 was expressed in carcinoma with an overall positivity rate of 67.76%, which was significantly higher than that in sarcomas (27.34%, P < 0.0001) and normal epithelial (5.18%, P < 0.0001) and fetal (2.67%, P < 0.0001) tissues. In epithelial tissues from 14 organs, the difference in HAb18G/CD147 expression between normal epithelium and the corresponding carcinoma was also significant ( P < 0.05 for each pair). This expression in carcinoma was also found at the mRNA level, suggesting transcriptional level regulation of HAb18G/CD147 expression. In a retrospective study of 106 patients with infiltrating ductal carcinoma of the breast, the level of HAb18G/CD147 expression was positively correlated with tumour recurrence/metastasis ( P = 0.0003) and negatively correlated with survival of breast cancer patients ( P = 0.002). Multivariable Cox regression analysis showed that HAb18G/CD147 was an independent prognostic factor. Conclusions: HAb18G/CD147 is significantly expressed in various cancers and appears to have prognostic significance, rendering it a possible cancer-associated biomarker for pathological diagnosis, prognostic evaluation, targeted therapy and radioimmunoimaging of a broad range of cancer types. [ABSTRACT FROM AUTHOR]

The article discusses a study which characterized the basigin isoforms and the function of basigin-3 to inhibit hepatocellular carcinoma proliferation and invasion.

CD 147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC) -associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers, However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8 Å resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal lgI domain, which are connected by a 5-residue flexible linker. This unique C2-L domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/heterooligomerizations and should represent a general architecture of other CD 147 family members. [ABSTRACT FROM AUTHOR]