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.

The selection of variable regions affects effector mechanisms of IgA antibodies against CD20

Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab and obinutuzumab are of human IgG1 isotype. Here, we investigated the impact of the variable regions of these three CD20 antibodies, when they were expressed as human IgA2 isotype variants. We observed more effective direct tumor cell killing by OBI-IgA2 compared to RTX- and OFA-IgA2, which was caspase-independent and required a functional cytoskeleton. Furthermore, IgA2 variants of all three antibodies triggered complement dependent cytotoxicity, with OBI-IgA2 being less effective than RTX- and OFA-IgA2. All three IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by PMN. Both effector mechanisms were significantly enhanced in the presence of a CD47 blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, OBI-IgA2 was consistently more potent than RTX- and OFA-IgA2 in triggering ADCC. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX- or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells, and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which is potentially relevant in the context of CD47-SIRPα blockade.

Novel oncolytic adenovirus expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitor activates multiple immune effector populations leading to enhanced tumor killing in vitro, in vivo and with patient-derived tumor organoids

BackgroundDespite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms.MethodsThe cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids.ResultsUsing various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations.ConclusionArming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.

Sterilizing immunity: New opportunities for rational TB vaccine design

Recent studies have revealed situations of high-level naturally acquired and vaccine-induced immunity against Mycobacterium tuberculosis in animal models along with examples of significantly protective immunization in humans. These discoveries offer immunologists new opportunities to define effector mechanisms that when triggered by appropriately engineered vaccines could end TB’s deadly reign.