Pantids for treatment of autoimmune disorders

#Autoimmune disorders are characterized by the gradual, and often progressive, decline of tolerogenesis and tolerogenic mechanisms that normally preclude adaptive immune responses to endogenous host proteins. During normal B and T cell development, autoreactive cells are eliminated in the bone marrow and thymus, respectively, creating “central tolerance” to host tissues and proteins. For B cells, expression of high-affinity B cell receptors (BCRs) to cell-surface proteins present in the bone marrow, the location of B cell development, results in apoptosis. Additionally, B cells that respond to ubiquitous soluble ligands are deactivated by anergy. For T cells, a similar process occurs in the thymus, the location of T cell development: T cells whose T cell receptor (TCR) responds with high-affinity to self-antigen peptides presented in MHC-I or MHC-II complexes are also deleted by apoptosis. For T cells with intermediate or low-affinity for said peptide-MHC complexes, these cells may develop into regulatory T cells (Tregs), which help maintain peripheral tolerance; alternatively, these cells may become anergic or undergo apoptosis.

#Peripheral tolerance refers to a suite of mechanisms that preclude adaptive immune responses to host proteins outside the central immune system. As afore-mentioned, these include centrally generated Treg cells, which help maintain peripheral tolerance by expressing immunosuppressive effectors in response to self-antigen peptide-MHC complexes. The mechanisms of Treg suppression are still being defined, but include the secretion of soluble immunosuppressive effectors and cell-contact-specific immunosuppressors. In the former mechanism, TGF-β, IL-10, adenosine (produced by CD39 and CD73), and IL-35 are secreted from Treg cells to create an immunosuppressive milieu that can prevent T and B cell activation, and create tolerogenic APCs1. In cell contact mechanisms, CTLA-4, PD-L1, LAG-3, membrane-bound TGF-β, and perforin and granzymes contribute to immunosuppression1. Also in the periphery, autoreactive T cells can be apoptosed or converted into peripheral Tregs by tolerogenic APCs, such as BTLA+ dendritic cells2. These peripheral Tregs (pTregs) contributed to peripheral tolerance through many of the mechanism described for central or thymic Tregs (cTregs or tTregs).

#An additional mechanism of peripheral tolerance is the general requirement for costimulation for T cell activation. When T cells engage their cognate antigen as peptide-MHC complex, there are two likely outcomes, depending on the presence of costimulation: in the presence of costimulatory agonist, such as CD80 or CD86 binding to T cell-expressed CD28, the T cell becomes activated, resulting in proliferation and the engagement of effector functions; in the alternate case, where costimulation is absent or when T cells receive inhibitory signals in lieu of or in combination with costimulatory signals, the T cells may undergo apoptosis, anergization, or conversion into pTreg. For B cells, a similar costimulation requirement exists for T cell-dependent B cell activation, wherein T cell-expressed CD40L must bind to B cell-expressed CD40 for B cell activation. While these canonical modalities of co-stimulation (e.g. CD28 and CD40) are the best described, additional co-stimulators and co-inhibitors have been recently elucidated: such receptors and their ligands, which cumulatively determine the outcome of antigen engagement, are referred to as immune checkpoint receptor or ligands: currently, these immunologic checkpoints include 15 signaling axes (FIG. 1).

#FIG. 1: Depiction of immunologic checkpoint receptors and their ligands. T cells receive a primary signal, depicted as “Signal 1,” when MHC-I or MHC-II:peptide complexes bind the T cell receptor (TCR). This signal primes the T cells for activation, anergy, or apoptosis. However, the fate of the T cell is ultimately determined by specific combinations of stimulatory and inhibitory immunological checkpoint receptor signaling, which can bias the T cell response towards one of these 3 outcomes.

#FIGS. 2 A and 2 B: Two instantiations of PantId technology. FIG. 2A provides a DNA fragment map of a PD-L1-FasL covalent molecular chimera. FIG. 2B provides maps of two DNA fragments separately encoding PD-L1 and FasL as molecular chimeras with cognate heterodimerization domains. In some embodiments the PantIds from FIG. 2B are co-transfected into mammalian cells after cloning into expression constructs for the production of PD-L1-CC-BN4:FasL-CC-AN4 heterodimers. This achieves the same therapeutic functionality of (A), but with simpler gene synthesis, cloning, and in vitro characterization.

#FIGS. 3 A and 3 B: Plasmid maps of PD-L1-FasL molecular chimera fragment, and cloned into lentivector pLenti-C-Myc-DDK-IRES-Puro. FIG. 3 A depicts a PD-L1-FasL molecular chimera fragment with terminal restriction sites, which allow cloning into pLenti-C-Myc-DDK-IRES-Puro. FIG. 3 B provides a plasmid map of a final pLenti-C-PD-L1-FasL-IRES-Puro vector, which would be used as both an expression vector, and as a lentivector for lentiviral transduction of producer cells.

#In one embodiment, this disclosure relates use of PantIda as therapeutics for the treatment of autoimmune diseases, characterized by autoreactive B cells which exhibit responsiveness to immunologic checkpoint receptors, or their ligands, or immunoregulatory cytokines.

#Although the mechanism of self-tolerance and autoimmunity is poorly understood for most autoimmune diseases, there are rare examples where the mechanism of initial tolerance is well characterized. For example, in Coxscackievirus B-associated myocarditis3 the initial viral infection is followed by inflammatory sequelae involving the myocardium and pericardium: this is associated with mononuclear cell infiltration, antibodies to cardiac actin and myosin, and associated CD4 T cells responses, which promote the clinical presentation of myocarditis3. In this example, Coxscackievirus-associated antigens molecularly mimic cardiac myosin and actin, and the resultant T and B cell responses continue in the absence of viral infection due to the capacity of cardiac myosin and actin to activate these autoreactive T and B cells. Similarly, in streptococcal-induced rheumatic heart disease, adaptive immune responses to streptococcal M protein cross-react with cardiac myosin and actin, resulting in a similar immunopathology4,5. Of note, these pathogen-associated autoimmune conditions are typically acute, and therefore, as recognized herein, other underlying predispositions towards autoimmunity likely coincide with such instigating stimuli to induce chronic clinical autoimmune diseases.

#As such, during the initial breakdown in self-tolerance, molecular mimicry between a pathogen's protein and a host protein can promote T and B cell reactivity to host proteins. More generally, the presence of alternate inflammatory stimuli in endogenous host tissues can result in aberrant T and B cell responses to these tissues. These inflammatory stimuli can lead to the expression of immunologic checkpoint co-stimulators that bypass one of the pivotal mechanisms of peripheral tolerance—the requirement for co-stimulation. More broadly, the initial inflammatory state that leads to checkpoint co-stimulator expression is not necessarily pathogen-derived, and could be caused by commensal bacteria, tissue injury, radiation, or chemical exposure, which can promote inflammation through pathogen-associated molecular pattern receptors (PAMPs) or damage-associated molecular pattern receptors (DAMPs). Alternatively, exposures to haptens, which covalently couple to host proteins and render them immunogenic, could lead to autoimmune responses in the presence of co-stimulation.