PD-L1 ⁺ tumor cells, but not macrophages, are likely responsible for inhibiting tumor-specific T cells in early-stage human lung tumors.

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.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

Background Glioblastoma multiforme (GBM) is known for its high antigenic heterogeneity, which undermines the effectiveness of monospecific immunotherapies. Multivalent immunotherapeutic strategies that target multiple tumor antigens simultaneously could enhance clinical outcomes by preventing antigen-driven tumor escape mechanisms. Methods We describe novel trivalent antibodies, DNA-encoded tri-specific T-cell engagers (DTriTEs), targeting two GBM antigens, epidermal growth factor receptor variant III (EGFRvIII) and IL-13Rα2, and engaging T cells through CD3. We engineered three DTriTE constructs, each with a unique arrangement of the antigen-binding fragments within a single-chain sequence. We assessed the binding efficiency and cytotoxic activity of these DTriTEs in vitro on target cells expressing relevant antigens. In vivo efficacy was tested in immunocompromised mice, including a longitudinal expression study post-administration and a survival analysis in an NOD scid gamma (NSG)-K mouse model under a heterogeneous tumor burden. RNA sequencing of DTriTE-activated T cells was employed to identify the molecular pathways influenced by the treatment. The antitumor cytotoxicity of patient-derived immune cells was evaluated following stimulation by DTriTE to assess its potential effectiveness in a clinical setting. Results All DTriTE constructs demonstrated strong binding to EGFRvIII and IL-13Rα2-expressing cells, induced significant T cell-mediated cytotoxicity, and enhanced cytokine production (interferon-γ, tumor necrosis factor (TNF)-α, and interleukin(IL)-2). The lead construct, DT2035, sustained expression for over 105 days in vivo and exhibited elimination of tumor burden in a heterogeneous intracranial GBM model, outperforming monospecific antibody controls. In extended survival studies using the NSG-K model, DT2035 achieved a 67% survival rate over 120 days. RNA sequencing of DTriTE-activated T cells showed that DT2035 enhances genes linked to cytotoxicity, proliferation, and immunomodulation, reflecting potent immune activation. Finally, DT2035 effectively induced target-specific cytotoxicity in post-treatment peripheral blood mononuclear cells from patients with GBM, highlighting its potential for clinical effectiveness. Conclusions DTriTEs exhibit potent anti-tumor effects and durable in vivo activity, offering promising therapeutic potential against GBM. These findings support further development of such multivalent therapeutic strategies to improve treatment outcomes in GBM and potentially other antigenically heterogeneous tumors. The opportunity to advance such important therapies either through biologic delivery or direct in vivo nucleic acid production is compelling.

Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings.

Background Advanced clear cell renal cell carcinoma (ccRCC) is a prevalent kidney cancer for which long-term survival rates are abysmal, though immunotherapies are showing potential. Not yet clinically vetted are bispecific T cell engagers (BTEs) that activate T cell-mediated cancer killing through intercellular synapsing. Multiple BTE formats exist, however, with limited cross-characterizations to help optimize new drug design. Here, we developed BTEs to treat ccRCC by targeting carbonic anhydrase 9 (CA9) while characterizing the persistent BTE (PBTE) format and comparing it to a new format, the persistent multivalent T cell engager (PMTE). These antibody therapies against ccRCC are developed as both recombinant and synthetic DNA (synDNA) medicines. Methods Antibody formatting effects on binding kinetics were assessed by flow cytometry and intercellular synaptic strength assays while potency was tested using T-cell activation and cytotoxicity assays. Mouse models were used to study antibody plasma and tumor pharmacokinetics, as well as antitumor efficacy as both recombinant and synDNA medicines. Specifically, three models using ccRCC cell line xenografts and human donor T cells in immunodeficient mice were used to support this study. Results Compared with a first-generation BTE, we show that the PBTE reduced avidity, intercellular synaptic strength, cytotoxic potency by as much as 33-fold, and ultimately efficacy against ccRCC tumors in vivo. However, compared with the PBTE, we demonstrate that the PMTE improved cell avidity, restored intercellular synapses, augmented cytotoxic potency by 40-fold, improved tumor distribution pharmacokinetics by 2-fold, and recovered synDNA efficacy in mouse tumor models by 20-fold. All the while, the PMTE displayed a desirable half-life of 4 days in mice compared with the conventional BTE’s 2 hours. Conclusions With impressive efficacy, the CA9-targeted PMTE is a promising new therapy for advanced ccRCC, which can be effectively delivered through synDNA. The highly potent PMTE format itself is a promising new tool for future applications in the multispecific antibody space.