SAIL66, a next generation CLDN6-targeting T-cell engager, demonstrates potent antitumor efficacy through dual binding to CD3/CD137.

BACKGROUND: Ovarian cancer remains a formidable challenge in oncology, necessitating innovative therapeutic approaches. Claudin-6 (CLDN6), a member of the tight junction molecule CLDN family, exhibits negligible expression in healthy tissues but displays aberrant upregulation in various malignancies, including ovarian cancer. Although several therapeutic modalities targeting CLDN6 are currently under investigation, there is still a need for more potent therapeutic options. While T-cell engagers (TCEs) hold substantial promise as potent immunotherapeutic agents, their current efficacy and safety in terms of target antigen selection and T-cell exhaustion due to only CD3 stimulation without co-stimulation must be improved, particularly against solid tumors. To provide an efficacious treatment option for ovarian cancer, we generated SAIL66, a tri-specific antibody against CLDN6/CD3/CD137. METHODS: Using our proprietary next-generation TCE technology (Dual-Ig), SAIL66 was designed to bind to CLDN6 with one Fab and CD3/CD137 with the other, thereby activating T cells through CD3 activation and CD137 co-stimulation. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3. RESULTS: Despite the high similarity between CLDN6 and other CLDN family members, SAIL66 demonstrated high specificity for CLDN6, reducing the risk of off-target toxicity. In an in vitro co-culture assay with CLDN6-positive cancer cells, we confirmed that SAIL66 strongly activated the CD137 signal in the Jurkat reporter system, and preferentially induced activation of both CD4+ and CD8+ T cells isolated from human peripheral blood mononuclear cells compared to conventional TCEs. In vivo studies demonstrated that SAIL66 led to a more pronounced increase in intratumor T-cell infiltration and a decrease in exhausted T cells compared with conventional CLDN6 TCE by contribution of CD137 co-stimulation, resulting in better antitumor efficacy in tumor-bearing mouse models. CONCLUSION: Our data demonstrate that SAIL66, designed to engage CLDN6, CD3, and CD137, has the potential to enhance antitumor activity and provide a potent therapeutic option for patients with ovarian and other solid tumors expressing CLDN6. Clinical trials are currently underway to evaluate the safety and efficacy of SAIL66.

Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression.

Two classes of Human papillomaviruses (HPV) infect the anogenital track: high risk viruses that are associated with risk of cervical cancer and low risk types that drive development of benign lesions, such as condylomas. In the present study, we established quantitative transcriptional maps of the viral genome in clinical lesions associated with high risk HPV16 or low risk HPV6b. Marked qualitative and quantitative changes in the HPV16 transcriptome were associated with progression from low to high grade lesions. Specific transcripts encoding essential regulatory proteins such as E7, E2, E1^E4 and E5 were identified. We also identified intrinsic differences between the HPV6b-associated condyloma transcript map and that of the HPV16-associated low grade CIN specifically regarding promoter usage. Characterization and quantification of HPV transcripts in patient samples thus establish the impact of viral transcriptional regulation on the status of HPV-associated lesions and may therefore help in defining new biologically-relevant prognosis markers.

Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes.

The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively.

Human papillomavirus proteins as prospective therapeutic targets.

Human papillomaviruses (HPV) are the causative agents of a subset of cervical cancers that are associated with persistent viral infection. The HPV genome is an ∼8 kb circle of double-stranded DNA that encodes eight viral proteins, among which the products of the E6 and E7 open reading frames are recognized as being the primary HPV oncogenes. E6 and E7 are expressed in pre-malignant lesions as well as in cervical cancers; hence these proteins have been extensively studied as potential targets for HPV therapies and novel vaccines. Here we review the expression and functions of E6 and E7 in the viral vegetative cycle and in oncogenesis. We also explore the expression and functions of other HPV proteins, including those with oncogenic properties, and discuss the potential of these molecules as alternative therapeutic targets.