OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*

BACKGROUNDOX40/OX40 ligand (OX40/OX40L) and programmed death-1/programmed death ligand-1 (PD-1/PD-L1) costimulatory signals play important roles in T cell-induced immune responses. The aim of this study was to investigate the roles of OX40/OX40L and PD-1/PD-L1 costimulatory pathways in mouse islet allograft rejection.

METHODSLentiviral vectors containing OX40L siRNA sequences and an adenovirus vector containing the PD-L1 gene were constructed. The streptozotocin-induced model of diabetes was established in C57BL/6 (H-2(b)) mice. Diabetic C57BL/6 mice were randomly allocated into five groups: group 1, untreated control; group 2, Ad-EGFP treatment; group 3, Ad-PD-L1 treatment; group 4, OX40L-RNAi-LV treatment; group 5, OX40L-RNAi-LV combined with Ad-PD-L1 treatment. Lentiviral vector and the adenovirus vector were injected, singly or combined, into the caudal vein one day before islet transplantation. The islets of DBA/2 (H-2(d)) mice were transplanted into the renal subcapsular space of the diabetic recipients. Recipient blood glucose and the survival time of the allografts were monitored. Antigen-specific mixed lymphocyte reaction was also evaluated.

RESULTSThe recombinant lentiviral RNA interference vector OX40L-RNAi-LV reduced OX40L protein expression by 70%. The recombinant adenovirus vector Ad-PD-L1 increased PD-L1 protein expression in vivo in C57BL/6 recipient mice. Combined OX40L-RNAi-LV/Ad-PD-L1 treatment induced a synergistic protective effect in pancreatic islet allografts. Allograft survival time in the combined treatment group was (92.27±9.65) days, not only longer than that of the control ((6.51±0.27) days) and Ad-EGFP groups ((7.09±0.13) days) (P < 0.01), but also significantly longer than that of Ad-PD-L1 and OX40L-RNAi-LV single treatment groups ((40.64±3.95) days and (55.14±5.48) days respectively, P < 0.01). The blood glucose concentration of recipient mice in the combined treatment group was also stable and kept within the normal range. Flow cytometry analysis showed that combined OX40L-RNAi-LV/Ad-PD-L1 treatment significantly decreased proliferation in an antigen-specific mixed lymphocyte reaction. After donor DBA/2 lymphocyte stimulation, 89.71% of lymphocytes from recipient combination treatment C57BL/6 mice were not split and proliferated. In contrast, after stimulation with third party Lewis rat lymphocytes, only 45.84% lymphocytes of C57BL/6 mice were not split and proliferated.

CONCLUSIONSThis study demonstrates the successful construction of the recombinant lentivirus vector OX40L-RNAi-LV and adenovirus vector Ad-PD-L1 for the blockade of OX40/OX40L and activation of PD-1/PD-L1 costimulatory pathways simultaneously in pancreatic islet allografts in diabetic mice. Combination therapy with these two vectors resulted in inhibition of T cell activation, synergistically prolonging the survival time of pancreatic islet allografts.

Animals ; B7-H1 Antigen ; genetics ; metabolism ; Graft Rejection ; genetics ; prevention & control ; Islets of Langerhans Transplantation ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; OX40 Ligand ; genetics ; metabolism ; Receptors, OX40 ; genetics ; metabolism ; Transplantation, Homologous

BACKGROUNDAcute rejection remains an important cause of renal allograft dysfunction and the need for accurate diagnosis is essential to successfully treat transplant recipients. The purpose of this study was to determine the costimulatory molecules OX40 and OX40L messenger RNA (mRNA) levels in peripheral blood mononuclear cells (PBMCs) to predict acute renal transplant rejection.

METHODSThe whole blood samples from 20 recipients with biopsy-confirmed acute rejection (rejection group), 20 recipients with stable graft function and normal biopsy results (stable group) after kidney transplantation, and 20 healthy volunteers (control group) were collected. The mRNA levels of OX40 and OX40L were analyzed with TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR). The association of OX40 and OX40L mRNA levels with disease severity was investigated.

RESULTSThere was no significant difference of OX40, OX40L mRNA levels in PBMCs between the stable group and control group (P > 0.05). The levels of OX40 and OX40L mRNA were significantly higher in the rejection group than in the control group (P < 0.01 and P < 0.05, respectively). Non-significantly higher OX40L mRNA and significantly higher OX40 mRNA in PBMCs were observed in subjects in the rejection group compared with the stable group (P > 0.05 and P < 0.01, respectively). Receiver operating characteristic (ROC) curve analysis demonstrated that OX40 mRNA levels could discriminate recipients who subsequently suffered acute allograft rejection (area under the curve, 0.908). OX40 and OX40L mRNA levels did not significantly correlate with serum creatinine levels in the rejection group (P > 0.05). Levels of OX40 mRNA after anti-rejection therapy were lower than those at the time of protocol biopsy in the rejection group (P < 0.05).

CONCLUSIONOur data suggest that measurement of OX40 mRNA levels after transplant might offer a noninvasive means for recognizing recipients at risk of acute renal allograft rejection.

Adult ; Biomarkers ; blood ; Female ; Graft Rejection ; blood ; diagnosis ; Humans ; Kidney Transplantation ; adverse effects ; Male ; Middle Aged ; OX40 Ligand ; genetics ; RNA, Messenger ; blood ; ROC Curve ; Receptors, OX40 ; genetics ; Transplantation, Homologous

Objective To explore the expressions and co-relationship of immune factors forkhead box p3 (FoxP3),chemokine (C-C motif) ligand 22 (CCL22),tumor necrosis factor receptor superfamily member 40(OX40),and SMAD family member 3 (Smad3) in cervical carcinoma and investigate their immunomodulatory roles in cervical carcinogenesis.Methods Totally 30 cases of cervical carcinoma with adjacent tissues and 20 cases of normal cervix were collected in this study. FoxP3,CCL22,OX40,and Smad3 mRNA expressions were detected by real-time polymerase chain reaction (RT-PCR). Results Compared to normal cervix,the expression levels of FoxP3 and CCL22 mRNA were elevated in neoplastic foci(P=0.000,P=0.002) and tumor periphery (P=0.048,P=0.040).The mRNAs increased modestly in high-grade squamous cell carcinoma focal(P=0.019,P=0.020) and periphery tissue (P=0.023,P=0.031) in comparison with low-grade squamous cell carcinoma. The expression levels of OX40 and Smad3 mRNA were significantly lower in neoplastic foci(P=0.000,P=0.015) than normal cervix. Compared to low-grade squamous cell carcinoma focal and periphery tissue,the mRNAs decreased moderately in high-grade squamous cell carcinoma(P=0.018,P=0.030; P=0.027,P=0.014). In both neoplastic foci and tumor periphery,the mRNA expression level of CCL22 was positively correlated with FoxP3 (r=0.353,P=0.000; r=0.307,P=0.000) but negatively correlated with OX40 (r=-0.288,P=0.031; r=-0.263,P=0.037),while OX40 was positively correlated with Smad3 (r=0.384,P=0.002;r=0.288,P=0.023). The mRNA expressions of FoxP3 and CCL22 were increased in foci and pericarcinous tissues (P=0.024,P=0.039; P=0.032,P=0.034) while Smad3 was decreased in neoplastic foci (P=0.017) in contrast to HPV negative corresponding group. Conclusion FoxP3 and CCL22 expressions increase while OX40 and Smad3 expression decrease at mRNA level in the microenvironment of cervical cancer,which may be associated with such immunological model that the immunosuppressive roles of FoxP3 and CCL22 enhance while the immunity-boosting roles of OX40 and Smad3 are impeded,contributing to the deterioration of immune disequilibrium in local site and cervical cancer carcinogenesis.
Carcinoma, Squamous Cell ; immunology ; Chemokine CCL22 ; metabolism ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, OX40 ; metabolism ; Smad3 Protein ; metabolism ; Uterine Cervical Neoplasms ; immunology

OBJECTIVE: To investigate the role of OX40 and Bcl-2 in allergic rhinitis(AR). METHOD: Twenty-three subjects of allergic rhinitis as experimental group were collected and 20 subjects of deflection of nasal septum as control group. OX40 and Bcl-2 expression were examined in biopsy specimens of AR patients and nasal septum patients by immunohistochemistry respectively. The relationship between OX40 and Bcl-2 expression was analyzed. RESULT: OX40 and Bcl-2 expression increased in the AR patients. There were positive staining not only in CD4+ T cells,but also in vascular endothelial cell, epithelial cell and glandular epithelium cell (P < 0.01). Compared with control group, the expression of OX40 (0.239 4 +/- 0.033 5) and Bcl-2 (0.237 3 +/- 0.042 1) were significantly higher than that in control group (P < 0.01). The OX40 expression was closely related to Bcl-2 expression (r = 0.869 0, P < 0.05). CONCLUSION The abnormal expression of costimulatory molecule OX40 is well evidenced in AR. Bcl-2 expression is likely mediated by OX40 signal pathway.
Adolescent ; Adult ; Biopsy ; Case-Control Studies ; Female ; Humans ; Middle Aged ; Nasal Mucosa ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Receptors, OX40 ; metabolism ; Rhinitis, Allergic, Perennial ; metabolism ; pathology ; Young Adult

OBJECTIVETo study the association of the OX40 gene rs2298212G/A polymorphism with coronary atherosclerotic disease (CAD) in Chinese Han population.

METHODSFive hundred and thirty six CAD patients and 544 age and ethnic matched controls of Chinese Han population were recruited from Qilu Hospital, Shandong University. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the selected single nucleotide polymorphism. Distributions of genotypic and allelic frequencies were analyzed by Chi-square test.

RESULTSThe distribution of genotypic and allelic frequencies have no significant differences between the CAD cases and controls(P> 0.05), even after adjusting for age, gender, body mass index, systolic blood pressure, diastolic blood pressure, glucose, total cholesterol, and triglyceride. However, when substratification analysis of the involved coronary artery vessels was performed, significant difference was found between single-vessel and triple-vessel (P= 0.02, OR = 1.56, 95% CI: 1.08-2.26) involvement.

CONCLUSIONThe rs2298212G/A polymorphism in OX40 gene may be associated with the severity of coronary atherosclerotic disease.

Asian Continental Ancestry Group ; ethnology ; genetics ; Base Sequence ; Coronary Artery Disease ; genetics ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Receptors, OX40 ; genetics

OBJECTIVE: To examine the expression of mRNA of Oxford 40(OX40) and Oxford 40 ligand(OX40L) in the sciatic nerve, spleen, peripheral blood mononuclear cells and lymph nodes of experimental allegic neuritis (EAN). METHODS: Thirty-six Lewis rats were randomly assigned into an EAN group and a CFA group. The rats were sacrificed on 9th, 17th, and 26th day after immunization. OX40 and OX40L mRNA was detected by reverse transcription polymerase chain reaction in the spleen, sciatic nerves, peripheral blood mononuclear cells and lymphonodes. RESULTS: The peak of clinical course came on 17th day after the immunization in EAN. The mRNA expression of OX40/OX40L was higher on 8th day and 17th day than that on 26th day after the immunization (P<0.05). There was significant difference between the EAN group and the CFA group at the 3 time points (P<0.05); rats in the CFA group didn't have any clinical manifestations. The mRNA expression of OX40 and OX40L in the EAN group raised in the sciatic nerves and lymph nodes at the above 3 time points (P<0.05). Weak expression was seen in the peripheral blood mononuclear cells. CONCLUSION OX40 and OX40L may play a role in the pathogenesis of experimental allegic neuritis.
Animals ; Female ; Membrane Glycoproteins ; genetics ; metabolism ; Neuritis, Autoimmune, Experimental ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred Lew ; Receptors, OX40 ; genetics ; metabolism ; Sciatic Nerve ; metabolism ; Tumor Necrosis Factors ; genetics ; metabolism