Aaron Prodeus

Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel Therapeutic Strategy for the Treatment of Disseminated Cancers

Blocking the immunoinhibitory PD-1:PD-L1 pathway using monoclonal antibodies has led to dramatic clinical responses by reversing tumor immune evasion and provoking robust and durable antitumor responses. Anti-PD-1 antibodies have now been approved for the treatment of melanoma, and are being clinically tested in a number of other tumor types as both a monotherapy and as part of combination regimens. Here, we report the development of DNA aptamers as synthetic, nonimmunogenic antibody mimics, which bind specifically to the murine extracellular domain of PD-1 and block the PD-1:PD-L1 interaction. One such aptamer, MP7, functionally inhibits the PD-L1-mediated suppression of IL-2 secretion in primary T-cells. A PEGylated form of MP7 retains the ability to block the PD-1:PD-L1 interaction, and significantly suppresses the growth of PD-L1+ colon carcinoma cells in vivo with a potency equivalent to an antagonistic anti-PD-1 antibody. Importantly, the anti-PD-1 DNA aptamer treatment was not associated with off-target TLR-9-related immune responses. Due to the inherent advantages of aptamers including their lack of immunogenicity, low cost, long shelf life, and ease of synthesis, PD-1 antagonistic aptamers may represent an attractive alternative over antibody-based anti PD-1 therapeutics.

p53 oligomerization status modulates cell fate decisions between growth, arrest and apoptosis

ABSTRACT Mutations in the oligomerization domain of p53 are genetically linked to cancer susceptibility in Li-Fraumeni Syndrome. These mutations typically alter the oligomeric state of p53 and impair its transcriptional activity. Activation of p53 through tetramerization is required for its tumor suppressive function by inducing transcriptional programs that lead to cell fate decisions such as cell cycle arrest or apoptosis. How p53 chooses between these cell fate outcomes remains unclear. Here, we use 5 oligomeric variants of p53, including 2 novel p53 constructs, that yield either monomeric, dimeric or tetrameric forms of p53 and demonstrate that they induce distinct cellular activities and gene expression profiles that lead to different cell fate outcomes. We report that dimeric p53 variants are cytostatic and can arrest cell growth, but lack the ability to trigger apoptosis in p53-null cells. In contrast, p53 tetramers induce rapid apoptosis and cell growth arrest, while a monomeric variant is functionally inactive, supporting cell growth. In particular, the expression of pro-arrest CDKN1A and pro-apoptotic P53AIP1 genes are important cell fate determinants that are differentially regulated by the oligomeric state of p53. This study suggests that the most abundant oligomeric species of p53 present in resting cells, namely p53 dimers, neither promote cell growth or cell death and that shifting the oligomeric state equilibrium of p53 in cells toward monomers or tetramers is a key parameter in p53-based cell fate decisions.

Induction of antigen-specific TH9 immunity accompanied by mast cell activation blocks tumor cell engraftment

The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen‐specific TH9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV‐like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA‐specific TH9 response, wherein IL‐9 secreting TH cells act in concert with CEA N domain‐specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3‐dsRNA complex formation led to a reduction in vaccine‐imparted protection and a shift in the resulting immune response toward a TH2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.

Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2

Angiopoietin‐1 (Ang1) activation of Tie2 receptors on endothelial cells (ECs) reduces adhesion by tumor cells (TCs) and limits junctional permeability to TC diapedesis. We hypothesized that systemic therapy with Vasculotide (VT)—a purported Ang1 mimetic, Tie2 agonist—can reduce the extravasation of potentially metastatic circulating TCs by similarly stabilizing the host vasculature. In vitro, VT and Ang1 treatments impeded endothelial hypermeability and the transendothelial migration of MDA‐MB‐231∙LM2‐4 (breast), HT29 (colon), or SN12 (renal) cancer cells to varying degrees. In mice, VT treatment inhibited the transit of TCs through the pulmonary endothelium, but not the hepatic or lymphatic endothelium. In the in vivo LM2‐4 model, VT monotherapy had no effect on primary tumors, but significantly delayed distant metastatic dissemination to the lungs. In the post‐surgical adjuvant treatment setting, VT therapeutically complemented sunitinib therapy, an anti‐angiogenic tyrosine kinase inhibitor which limited the local growth of residual disease. Unexpectedly, detailed investigations into the putative mechanism of action of VT revealed no evidence of Tie2 agonism or Tie2 binding; alternative mechanisms have yet to be determined.

Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That Prolong Allogeneic Skin Graft Survival.

CD200R1 expressed on the surface of myeloid and lymphoid cells delivers immune inhibitory signals to modulate inflammation when engaged with its ligand CD200. Signalling through CD200/CD200R1 has been implicated in a number of immune-related diseases including allergy, infection, cancer and transplantation, as well as several autoimmune disorders including arthritis, systemic lupus erythematosus, and multiple sclerosis. We report the development and characterization of DNA aptamers, which bind to murine CD200R1 and act as potent signalling molecules in the absence of exogenous CD200. These agonistic aptamers suppress cytotoxic T-lymphocyte induction in 5-day allogeneic mixed leukocyte culture and induce rapid phosphorylation of the CD200R1 cytoplasmic tail thereby initiating immune inhibitory signalling. PEGylated conjugates of these aptamers show significant in vivo immunosuppression and enhance survival of allogeneic skin grafts as effectively as soluble CD200Fc. As DNA aptamers exhibit inherent advantages over conventional protein-based therapeutics including low immunogenicity, ease of synthesis, low cost, and long shelf life, such CD200R1 agonistic aptamers may emerge as useful and safe nonsteroidal anti-inflammatory therapeutic agents.

Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis

VEGF‐A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF‐induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF‐induced blood vessel formation ex vivo. SPARC decreases VEGF‐induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and /or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 104 M‐1s‐1)with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10‐3 s‐1). Only domain III of SPARC binds VEGF, exhibiting a 15‐fold higher affinity than full‐length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.— Cydzik, M., Abdul‐Wahid, A., Park, S., Bourdeau, A., Bowden, K., Prodeus, A., Kollara, A., Brown, T. J., Ringuette, M. J., Gariépy, J. Slow binding kinetics of secreted protein, acidic, rich in cysteine‐VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis. FASEB J. 29, 3493‐3505 (2015). www.fasebj.org

VISTA.COMP — an engineered checkpoint receptor agonist that potently suppresses T cell–mediated immune responses

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped, largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However, this effect required its immobilization on a solid surface, suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein, we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc, VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore, we show that VISTA.COMP, but not VISTA-Fc, functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively, we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses.

Association Between the Oligomeric Status of p53 and Clinical Outcomes in Li-Fraumeni Syndrome

Li-Fraumeni syndrome (LFS) is a rare hereditary cancer disorder with highly variable clinical outcomes that results from germline mutations in the TP53 gene. Here we report that the quaternary structure of p53 is an important factor affecting cellular functions and the clinical outcomes of LFS patients (n = 87). Specifically, carriers of monomeric p53 mutants (n = 56) exhibited complete penetrance, with a 2.11-fold greater risk of cancer-related death (95% confidence interval [CI] = 1.07 to 4.30) and a statistically significantly lower median survival age as compared with carriers of multimeric (dimeric or tetrameric, n = 31) p53 mutants (33 years, 95% CI = 30 to 50, vs 51 years, 95% CI = 40 to NA, respectively, two-sided P = .03), who presented incomplete penetrance. Cellular functional assays using p53-null H1299 cells expressing clinically relevant p53 mutants confirmed that the cellular effects observed upon loss of p53 oligomerization are associated with clinical outcomes of LFS patients. The association between p53 oligomeric state and clinical phenotype suggests that TP53 mutations are not all equivalent and supports the implementation of new genotype-adapted guidelines for the management of LFS patients with TP53 mutations in the oligomerization domain.

A Synthetic Cross-Species CD200R1 Agonist Suppresses Inflammatory Immune Responses In Vivo

Functional aptamers displaying agonistic or antagonistic properties are showing great promise as modulators of immune responses. Here, we report the development of a polyethylene glycol-modified (PEGylated) DNA aptamer as a cross-species (murine and human) CD200R1 agonist that modulates inflammatory responses in vivo. Specifically, DNA aptamers were discovered by performing independent SELEX searches on recombinant murine and human CD200R1. Aptamer motifs identified by next generation sequencing (NGS) were subsequently compared, leading to the discovery of motifs common to both targets. The CD200R1 DNA aptamer CCS13 displayed the highest agonistic activity toward CD200R1 in terms of suppressing the induction of cytotoxic T-lymphocytes (CTLs) in both human and murine allogeneic-mixed lymphocyte cultures (allo-MLCs). A 20-kDa polyethylene glycol (PEG) chain was covalently attached to the 5′ end of this aptamer, and the resulting conjugate was shown to block inflammatory responses in murine models of skin graft rejection and house-dust-mite-induced allergic airway inflammation. Importantly, this agonistic aptamer does not suppress CTL induction in 5-day allo-MLCs with responder cells derived from CD200R1−/− mice, indicating that its mode of action is directly linked to CD200R1 activation. This study suggests that one can derive agonistic DNA aptamers that can be verified as immuno-modulators in murine models with outcomes potentially translatable to the treatment of human conditions.

Survival in males with glioma and gastric adenocarcinoma correlates with mutant p53 residual transcriptional activity

BACKGROUND There is currently no clinical distinction between different TP53 mutations, despite increasing evidence that not all mutations have equally deleterious effects on the activity of the encoded tumor suppressor protein p53. The objective of this study was to determine whether these biological differences have clinical significance. METHODS This retrospective cohort analysis included 2,074 patients with sporadic TP53 mutations (403 unique mutations) and 1,049 germline TP53 mutation carriers (188 unique mutations). Survival was projected by stratifying patients according to their p53 mutant-specific residual transcriptional activity scores. RESULTS Pan-cancer survival analyses revealed a strong association between increased mutant p53 residual activity and improved survival in males with glioma and gastric adenocarcinoma (P = 0.002 and P = 0.02) that was not present in the female cohorts (P = 0.16 and P = 0.50). Male glioma and gastric cancer patients with TP53 mutations resulting in >5% transcriptional activity had 3.1-fold (95% CI, 2.4-3.8; P = 0.002; multivariate analysis hazard ratio [HR]) and 4.6-fold (95% CI, 3.7-5.6; P = 0.001; multivariate analysis HR) lower risk of death as compared with patients harboring inactive (0% activity) p53 mutants. The correlation between mutant p53 residual activity with survival was recapitulated in the dataset of germline TP53 mutation carriers (HR = 3.0, 95% CI, 2.7-3.4, P < 0.001 [females]; HR = 2.2, 95% CI, 1.8-2.6, P < 0.001 [males]), where brain and gastric tumors were more common among males (P < 0.001 and P = 0.001, respectively). CONCLUSION The retention of mutant p53 transcriptional activity prognosticates superior survival for men with glioma and gastric adenocarcinoma harboring sporadic TP53 mutations. Among germline TP53 mutation carriers, increased residual transcriptional activity is correlated with prolonged lifetime cancer survival and delayed tumor onset, and males are more prone to develop brain and gastric tumors. FUNDING Canadian Institutes of Health Research (no. 148556).