Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma.

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.

Siglec-7 glyco-immune binding mAbs or NK cell engager biologics induce potent antitumor immunity against ovarian cancers.

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.

DNA immunotherapy targeting BARF1 induces potent anti-tumor responses against Epstein-Barr-virus-associated carcinomas.

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.

In vivo DNA-launched bispecific T cell engager targeting IL-13Rα2 controls tumor growth in an animal model of glioblastoma multiforme.

Glioblastoma is an aggressive tumor with poor survival rates. Bispecific T cell engagers (BTEs) against different cancers are in various stages of clinical development. Toxicity resulting from cytokine release syndrome and the short half-life of BTEs, which necessitates continuous infusion, complicating delivery and increasing costs, are major challenges in the field. Here we describe the development of in vivo DNA-launched BTEs (dBTEs) with highly focused targeting of interleukin-13 receptor α2 (IL-13Rα2), a glioblastoma cell-surface target. We developed 4 BTEs targeting 2 epitopes of IL-13Rα2 and studied how heavy-light chain orientation affects BTE function. The dBTEs induced T cell activation, cytokine production, and tumor cytolysis in the presence of IL-13Rα2+ tumor cells, but we observed unique patterns of immune activation. We found a strong correlation between granzyme B secretion and dBTE-induced cytolysis of specific and nonspecific tumors. We down-selected dBTE PB01-forward based on lower cytokine induction profile and highest activation specificity. In vivo, dBTE PB01-forward demonstrated an improved half-life versus intravenous recombinant BTE delivery. In an orthotopic glioblastoma model, dBTE PB01-forward controlled tumor growth, improving animal survival, supporting the hypothesis that the blood-brain barrier does not affect the function of systemically delivered dBTE. Further study of PB01-forward for targeting glioblastoma and other IL-13Rα2+ cancers is warranted.

A mAb against surface-expressed FSHR engineered to engage adaptive immunity for ovarian cancer immunotherapy.

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 EC50s 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.

A synDNA vaccine delivering neoAg collections controls heterogenous, multifocal murine lung and ovarian tumors via robust T cell generation.

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.

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.

Origin and Role of a Subset of Tumor-Associated Neutrophils with Antigen-Presenting Cell Features in Early-Stage Human Lung Cancer.

Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b(+)CD15(hi)CD10(-)CD16(low) immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor T cell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer.

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer.

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.