The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

Ovarian cancer (OC) is a lethal gynecologic malignancy, with modest responses to CPI. Engagement of additional immune arms, such as NK cells, may be of value. We focused on Siglec-7 as a surface antigen for engaging this population. Human antibodies against Siglec-7 were developed and characterized. Coculture of OC cells with PBMCs/NKs and Siglec-7 binding antibodies showed NK-mediated killing of OC lines. Anti–Siglec-7 mAb (DB7.2) enhanced survival in OC-challenged mice. In addition, the combination of DB7.2 and anti–PD-1 demonstrated further improved OC killing in vitro. To use Siglec-7 engagement as an OC-specific strategy, we engineered an NK cell engager (NKCE) to simultaneously engage NK cells through Siglec-7, and OC targets through FSHR. The NKCE demonstrated robust in vitro killing of FSHR ⁺ OC, controlled tumors, and improved survival in OC-challenged mice. These studies support additional investigation of the Siglec-7 targeting approaches as important tools for OC and other recalcitrant cancers.

Specific antibody therapy, including mAbs and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production, thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery, and in vivo production would be an important advance. Here we describe the development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2dMAb) and delivered it into animals through adaptive electroporation. HER2dMAb was efficiently expressed in vitro and in vivo, reaching levels of 50 μg/ml in mouse sera. Mechanistically, HER2dMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2dMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2dMAb single-chain variable fragment portion to engineer a DNA-encoded BiTE (DBiTE). This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate an approach to generate DBiTEs in vivo, which represent promising immunotherapies for HER2 ⁺ tumors, including ovarian and potentially other cancers.

Despite advances in ovarian cancer (OC) therapy, recurrent OC remains a poor-prognosis disease. Because of the close interaction between OC cells and the tumor microenvironment (TME), it is important to develop strategies that target tumor cells and engage components of the TME. A major obstacle in the development of OC therapies is the identification of targets with expression limited to tumor surface to avoid off-target interactions. The follicle-stimulating hormone receptor (FSHR) has selective expression on ovarian granulosa cells and is expressed on 50%–70% of serous OCs. We generated mAbs targeting the external domain of FSHR using in vivo–expressed FSHR vector. By high-throughput flow analysis, we identified multiple clones and downselected D2AP11, a potent FSHR surface–targeted mAb. D2AP11 identifies important OC cell lines derived from tumors with different mutations, including BRCA1/2, and lines resistant to a wide range of therapies. We used D2AP11 to develop a bispecific T cell engager. In vitro addition of PBMCs and T cells to D2AP11-TCE induced specific and potent killing of different genetic and immune escape OC lines, with EC ₅₀ s in the ng/ml range, and attenuated tumor burden in OC-challenged mouse models. These studies demonstrate the potential utility of biologics targeting FSHR for OC and perhaps other FSHR-positive cancers.

Latent Epstein-Barr virus (EBV) infection is associated with several types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV malignancies, suggesting that additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV⁺ nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC). BARF1 antigen can transform human epithelial cells in vivo. BARF1-specific antibodies and cytotoxic T cells were detected in some EBV⁺ NPC patients. However, BARF1 has not been evaluated as an antigen in the context of therapeutic immunization. Its possible importance in this context is unclear. Here, we developed a synthetic-DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and T cell responses in vivo. Immunization with pBARF1 plasmid impacted tumor progression through the induction of CD8⁺ T cells in novel BARF1⁺ carcinoma models. Using an in vivo imaging system, we observed that pBARF1-immunized animals rapidly cleared cancer cells. We demonstrated that pBARF1 can induce antigen-specific immune responses that can impact cancer progression. Further study of this immune target is likely important as part of therapeutic approaches for EBV⁺ malignancies.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen that causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion of sows and the manifestation of respiratory diseases in piglets. PRRSV strains are categorized into two distinct genotypes: PRRSV–1 and PRRSV–2. PRRSV–2 can be further classified into several lineages, including sub–lineage 1.8 (NADC30–like), sub–lineage 1.5 (NADC34–like), lineage 8 (HP–PRRSV–like), lineage 5 (VR–2332–like), and lineage 3 (QYYZ–like), all of which are prevalent in China. In order to identify PRRSV–1 and PRRSV–2, two primer–probe combinations were designed, targeting the M gene. In order to further differentiate the five lineages of PRRSV–2, another five primer–probe combinations were designed, targeting the Nsp2 gene. A TaqMan–based multiplex RT–qPCR assay was subsequently developed, integrating the aforementioned seven sets into two primer pools. Following the optimization of primer concentration and annealing temperature, a comprehensive evaluation was conducted to assess the assay’s amplification efficiency, specificity, repeatability, and sensitivity. The developed multiplex RT–qPCR method exhibited excellent repeatability, with coefficients of variation (CVs) less than 2.12%. The detection limits for all seven targets were found to be less than 5 copies/μL. Ultimately, the method was utilized for the detection of a total of 1009 clinical samples, with a PRRSV–positive rate of 7.63% (77/1009). Specifically, the reference method was utilized to further confirm the status of the 77 PRRSV–positive samples and another 27 samples suspected of PRRSV infection. The sensitivity of the method was 97.40% (75/77), and the specificity was 96.30% (26/27), resulting in an overall coincidence rate of 97.12% (101/104). All the PRRSV–positive samples were typed as NADC30–like strains, and the accuracy of this typing was further confirmed by Sanger sequencing. In conclusion, A one–step multiplex RT–qPCR method was successfully constructed, evaluated, and applied to detect clinical samples. The assay provides an easy–to–operate, time–saving, and highly efficient way for the quick identification of PRRSV and simultaneous detection of five PRRSV–2 lineages prevalent in China. The method could offer guidance for PRRSV prevention and control measures.