Aws Abdul-Wahid

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.

The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP–protein cargos to the cytosol of eukaryotic cells

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.

Blocking the attachment of cancer cells in vivo with DNA aptamers displaying anti-adhesive properties against the carcinoembryonic antigen

The formation of metastatic foci occurs through a series of cellular events, initiated by the attachment and aggregation of cancer cells leading to the establishment of micrometastases. We report the derivation of synthetic DNA aptamers bearing anti‐adhesive properties directed at cancer cells expressing the carcinoembryonic antigen (CEA). Two DNA aptamers targeting the homotypic and heterotypic IgV‐like binding domain of CEA were shown to block the cell adhesion properties of CEA, while not recognizing other IgV‐like domains of CEACAM family members that share strong sequence and structural homologies. More importantly, the pre‐treatment of CEA‐expressing tumour cells with these aptamers prior to their intraperitoneal implantation resulted in the prevention of peritoneal tumour foci formation. Taken together, these results highlight the effectiveness of targeting the cell adhesion properties of cancer cells with aptamers in preventing tumour implantation.

An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

BackgroundFew treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM) allowing the toxin variant to selectively target and kill human melanoma cells.ResultsSLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1AIYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice.ConclusionsThese results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

A focused immune response targeting the homotypic binding domain of the carcinoembryonic antigen blocks the establishment of tumor foci in vivo

Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA‐expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self‐form of the CEA Ig V‐like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA‐mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody‐dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients.

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.

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.

The carcinoembryonic antigen IgV-like N domain plays a critical role in the implantation of metastatic tumor cells

The human carcinoembryonic antigen (CEA) is a cell adhesion molecule involved in both homotypic and heterotypic interactions. The aberrant overexpression of CEA on adenocarcinoma cells correlates with their increased metastatic potential. Yet, the mechanism(s) by which its adhesive properties can lead to the implantation of circulating tumor cells and expansion of metastatic foci remains to be established. In this study, we demonstrate that the IgV‐like N terminal domain of CEA directly participates in the implantation of cancer cells through its homotypic and heterotypic binding properties. Specifically, we determined that the recombinant N terminal domain of CEA directly binds to fibronectin (Fn) with a dissociation constant in the nanomolar range (KD 16 ± 3 nM) and interacts with itself (KD 100 ± 17 nM) and more tightly to the IgC‐like A3 domain (KD 18 ± 3 nM). Disruption of these molecular associations through the addition of antibodies specific to the CEA N or A3B3 domains, or by adding soluble recombinant forms of the CEA N, A3 or A3B3 domains or a peptide corresponding to residues 108–115 of CEA resulted in the inhibition of CEA‐mediated intercellular aggregation and adherence events in vitro. Finally, pretreating CEA‐expressing murine colonic carcinoma cells (MC38.CEA) with rCEA N, A3 or A3B3 modules blocked their implantation and the establishment of tumor foci in vivo. Together, these results suggest a new mechanistic insight into how the CEA IgV‐like N domain participates in cellular events that can have a macroscopic impact in terms of cancer progression and metastasis.

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.