Complement-Mediated Selective Tumor Cell Lysis Enabled by Bi-Functional RNA Aptamers

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, the powerful effector mechanisms of the immune response can be conscripted to treat cancer. To implement this strategy, we have been developing aptamer-derived molecular adaptors to invoke synthetic immune responses against cancer cells. Here we describe multi-valent aptamers that simultaneously bind target molecules on the surface of cancer cells and an activated complement protein, which would tag the target molecules and their associated cells as “foreign” and trigger multiple effector mechanisms. Increased deposition of the complement proteins on the surface of cancer cells via aptamer binding to membrane targets could induce the formation of the membrane attack complex or cytotoxic degranulation by phagocytes and natural killer cells, thereby causing irreversible destruction of the targeted cells. Specifically, we designed and constructed a bi-functional aptamer linking EGFR and C3b/iC3b, and used it in a cell-based assay to cause lysis of MDA-MB-231 and BT-20 breast cancer cells, with either human or mouse serum as the source of complement factors.

Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.

Probiotic-guided CAR-T cells for universal solid tumor targeting

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of any singular therapy. One major challenge of tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of targetable antigens that are specifically and uniformly expressed on heterogenous solid tumors. In contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be readily engineered as antigen-independent platforms for therapeutic delivery. Bridging these approaches, we develop a platform of probiotic-guided CAR-T cells (ProCARs), in which T cells are engineered to sense synthetic antigens (SA) that are produced and released by tumor-colonizing probiotic bacteria. We demonstrate increased CAR-T cell activation and tumor-cell lysis when SAs anchor to components of the extracellular matrix. Moreover, we show that ProCARs are intratumorally activated by probiotically-delivered SAs, receive further stimulation from bacterial TLR agonists, and are safe and effective in multiple xenograft models. This approach repurposes tumor-colonizing bacteria as beacons that guide the activity of engineered T cells, and in turn builds the foundation for communities of living medicines.

Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing

Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissected PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identified Rnf31 and Vps4b as essential factors required for escaping CD8+ T cell-killing. Using murine PDA cells and human PDA organoids, we demonstrate that Rnf31 protects from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction. For Vps4b we found that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. Orthotopic transplantation of Rnf31- or Vps4b deficient pancreatic tumors, moreover, revealed increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth in mice. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

634 Production and testing of a novel bispecific nanobody construct targeting NK cells and EGFR expressing malignancies

BackgroundThe ability to kill tumor cells with an acceptable toxicity profile, makes Natural Killer (NK) cells promising assets for cancer therapy. However, strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment would likely increase the efficacy of NK cell-based therapies.MethodsIn this study, we show a novel bispecific nanobody-based construct (biVHH) targeting both CD16A (low-affinity Fc receptor: FcRγIIIA) on NK cells and EGFR on tumors of epithelial origins.ResultsHigher levels of NK cell activity and subsequent tumor cell lysis were found in vitro in the presence of the biVHH and were dependent on the expression of both CD16A and EGFR while they were independent of the KRAS mutational status of the tumor. Increased NK cell activity was found in NK cells derived from colorectal cancer (CRC) patients when co-cultured with the biVHH and EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the biVHH and autologous peripheral blood mononuclear cells or allogeneic NK cells.ConclusionsBased on our results, the bispecific CD16A and EGFR targeting VHH construct could be a useful tool in combination with various NK cell-based therapies.

COVD-18. POTENTIAL TO HARNESS SARS-COV-2 NEUROTROPISM IN THE DELIVERY OF ONCOLYTIC VIROTHERAPY FOR THE TREATMENT OF HIGH-GRADE GLIOMA

BACKGROUND High-grade gliomas (HGG) pose therapeutic challenges stemming from blood brain barrier, infiltrative growth, suppressed immune function, and tumor heterogeneity. Oncolytic viruses (OVs) are gaining traction for addressing these challenges. There is evidence that the SARS-CoV-2 glycoprotein spike binds the ACE-2 receptor in nasal epithelium and reaches the brainstem and thalamus via axonal transport through the olfactory pathway, making it an attractive candidate for OV therapy. Prior studies on chimerization of pathogenic virus-derived glycoprotein spikes with non-pathogenic strains exploit neurotropism of a wild-type virus while improving the safety profile of the resulting OV. We review, 1) the engineering of chimeric OVs used in the treatment of HGG; 2) potential for a novel chimeric virotherapy in which the SARS-CoV-2 glycoprotein spike can be used to deliver OV therapy intranasally; and 3) areas which warrant further investigation to develop this approach for clinical use. METHODS We performed an extensive review of chimeric OVs and specific modifications engineered to optimize safety and efficacy. Additionally, we assessed potential to use these principals to engineer the SARS-CoV-2 glycoprotein spike onto a non-pathogenic, replication competent virus to yield a novel chimeric for noninvasive, intranasal delivery. RESULTS Viruses with pathogenic properties in wild-type have been successfully used as components of OVs and have demonstrated potential in both preclinical and clinical trials. Outcomes show that despite wild-type virulence, notable toxicities were not observed in clinical trials, highlighting the potential of viral pseudotyping as a safe therapeutic approach. CONCLUSIONS The proposed method to utilize the SARS-CoV-2 glycoprotein in a novel chimeric poses advantages including 1) potential for non-invasive delivery, 2) therapy without need for maximal or uniform tumor coverage due to replication competence, 3) ability to reach infiltrative glioma cells, 4) potential to reach the brainstem, and 5) stimulation of host immunity through tumor cell lysis and antigen presentation