Induction of antigen-specific TH 9 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 TH 9 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 TH 9 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 TH 2 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.

Labeling Cell Surface Receptors with Ligand.BirA* Bispecifics.

BirA*, a mutant form of the biotinylating enzyme BirA, can nonspecifically biotinylate ε-amino groups on lysines of proteins. Based on the promiscuous labeling nature of BirA*, plasmids expressing fusion constructs of BirA* to a given ligand have been used to transfect eukaryotic cells, leading to the biotinylation of intracellular proteins interacting or in close proximity to such Ligand.BirA* constructs. Mass spectrometry performed on the recovered biotinylated partners allows one to map intracellular protein interactors, a technique known as BioID. In contrast, the expression and purification of recombinant Ligand.BirA* constructs could serve as a powerful tool for labeling and detecting cell surface receptors. Here, we report the design and expression of recombinant Affibody.BirA* constructs, ZEGFR:1907.BirA* and ZHER2:243.BirA*, as protein bispecifics able to biotinylate their respective receptors EGFR and HER2 on the surface of MDA-MB-231 (EGFR+, EpCaM+, and CD44+) and SK-OV-3 (HER2++, EGFR+, EpCaM+, and CD44+) cancer cells. These Affibody.BirA* constructs retain both their BirA* enzymatic activity as well as their receptor-binding function. Importantly, MDA-MB-231 and SK-OV-3 cells biotinylated with Affibody.BirA* constructs did label their receptors EGFR and HER2 but did not biotinylate irrelevant antigens such as EpCaM or CD44 present on the surface of both cell lines. Ligand.BirA* bispecifics may represent a promising class of agents to identify unknown receptors on cell surfaces.

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.