Research

RESOLUTION OF HIV LATENCY BY CAR T CELL SENTINELS

Antiretroviral therapy (ART) has proven highly effective in HIV treatment, lowering plasma viral loads to undetectable levels and preventing disease progression.  However, despite rendering HIV a mostly manageable rather than mortal disease, ART is limited by cost, potential long-term toxicity, and, importantly, it does not eliminate the latently infected reservoir of cells.  The necessity for the complete elimination of all HIV viral reservoirs highlights the need for new treatment approaches. 

A cure for HIV would require the elimination of the latently infected reservoir of host cells while simultaneously diminishing the acquisition of target cell infections, a process proven to be quite difficult to date.  One promising approach to combat HIV latency within infected individuals would be a single autologous self-sustaining curative that constantly scans the host, targeting and rapidly killing recently activated reservoir cells while simultaneously constitutively secreting functional concentrations of broadly neutralizing mono-, bi-, or tri-specific antibodies (bnAbs).  Thus, targeting and killing host cells that are switching from latent to productive infection while simultaneously neutralizing circulating free virus.  One such exciting approach is the use of CAR T cell immunotherapy.  As discussed above, CAR T cells have become important for cancer therapy, and we propose to (re)adapt this novel approach for HIV.  Indeed, adoptive immunotherapy was initially tested for HIV, and lessons learned from those studies led to rapid advances in the field, particularly for cancer treatment.  T cells transduced to express CD4 on their surface linked to activation signaling machinery were employed to target and kill infected host cells.  But this approach had substantial drawbacks, including a novel entry route for infection of CD8 T cells, shielding of the CD4-binding site, and overall lack of efficacy.  Most disappointingly, this approach only modestly lowered viremia and was eventually abandoned as a potential HIV therapy.  Yet, these initial clinical studies demonstrated the overall safety of administering retrovirally modified T cells to humans.  Moreover, it was demonstrated that the engineered T cells trafficked to known reservoirs of natural HIV infection (i.e., the mucosa) and showed surprisingly long persistence, with half-lives of >16 years.   Importantly, CAR T cells are especially suited to killing cells with very low antigen densities.  Thus, their self-sustaining nature and ability to traffic and surveil virtually the entire host are highly beneficial for controlling a chronic systemic pathogen such as HIV.  Utilizing secreted high-affinity anti-HIV bnAbs, which are remarkably effective across a wide swath of HIV strains, in conjunction with superior CAR designs, we propose to exploit both advancements to generate doubly immunotherapeutic improved dual CAR T cells.  These pioneering studies should provide new insights into how to access and control the reservoir of latently HIV-infected cells and, in combination with ART, may address the exigent goal of functional control of HIV that would permit the eventual cessation of lifelong ART.

CAR T CELLS TARGETING LONG-LIVED PLASMA AND PLASMACYTOID DENDRITIC CELLS

Immunotherapies are becoming attractive alternatives to surgical, chemo- and radiotherapies for cancer.  Gene therapy methods that “reteach” the immune system to recognize and eliminate tumor cells via chimeric antigen receptors (CARs) have produced exciting results by us and others in xenograft models.  CAR T cells are engineered by retroviral transduction of a patient’s own CD8 T cells endowing these cells with the ability to seek and destroy specific target cells.  These studies will use the CAR T cell approach but apply it to mouse models of Systemic Lupus Erythematosus (SLE) as proof of concept to demonstrate that CAR T cells can effectively eliminate potentially pathologic effector cells in autoimmunity. 

SLE, the prototypic systemic autoimmune disease, is a heterogeneous multi-organ autoimmune disease characterized by a plethora of autoantibodies to nuclear and cytoplasmic components, immune complex-mediated glomerulonephritis (GN), and early death.  Susceptibility is influenced by genetic, environmental, hormonal, and stochastic factors.  Autoantibodies typically arise before overt manifestations of disease, and high titers of anti-dsDNA are associated with greater severity.  Similar findings are observed in lupus-prone mice, and, importantly, passively administered anti-DNA mAbs can produce lupus-like immune complex kidney deposits.  Endosomal Toll-like receptors (TLRs: TLR3, TLR7, TLR9) in B cells, plasmacytoid dendritic cells (pDCs), and conventional DCs are also thought to play important roles in lupus development through the recognition of self-nucleic acids and related immune complexes as well as the release of type I IFNs.  The specific elimination of certain immune effector cells, specifically plasma cells and/or plasmacytoid dendritic cells, by BiFabs or CAR T cells may alleviate many lupus manifestations and/or ameliorate ongoing disease. 

B cells play a dual role in lupus by acting as antigen-presenters and producing copious amounts of autoantibodies, particularly following their maturation to plasma cells.  This dual nature was a prime consideration of the hypothesis that B cell depletion via Rituximab would be beneficial in SLE.  However, this treatment modality was mostly ineffective, and the failure was theorized to be partly due to the inability of Rituximab to ablate long-lived plasma cells (LLPCs).  Indeed, current SLE therapies are immunosuppressive and target many inflammatory cells as well as deplete short-lived plasma cells.  But these approaches are associated with significant toxicity, unwanted side effects, little efficacy, and high relapse rates.  Newer approaches specifically targeting LLPCs are highly warranted. 

Dendritic cells are divided into several subsets, including conventional DCs (cDCs) and pDCs, the latter thought to be the chief producers of type I IFNs.  pDCs are longer lived than cDCs and can be found in almost all peripheral organs, blood, and bone marrow.  They are productive antigen presenters and, as mentioned, elaborate large amounts of type I IFNs, particularly in response to viral challenge and immune complex-mediated TLR stimulation.  In SLE patients, pDCs are found in low numbers in the peripheral blood, but increased numbers are seen in target organs and cutaneous lesions.  Similar findings have been reported in Sjogren’s syndrome, RA, dermatomyositis, and psoriasis.  Newer strategies to specifically target pDCs, thus lowering type I IFN levels, is the avenue we intend to explore. 

We have proposed that removing LLPCs and/or pDCs by CAR T cells will benefit many autoantibody-driven autoimmune diseases, including SLE. 

OLIGODENDROCYTE PROGENITOR CELLS INDUCED TO MATURE AND REMYELINATE

Multiple Sclerosis (MS) is an autoimmune disease characterized by multi-focal demyelination of axons and neuronal dysfunction.  Its pathology is typified by immune cell infiltration, demyelination, and oligodendrocyte loss.  More severe progressive phases of MS are associated with inhibited differentiation of the oligodendrocyte progenitor cell (OPC) population, which generates mature oligodendrocytes required for axonal remyelination.  Evidence suggests that remyelination (and remission) is dependent upon the recruitment of OPCs to the demyelinated areas, followed by their differentiation into myelin-producing cells to rewrap damaged axons in myelin, restoring electrical conduction between neurons. 

Normally, remyelination in the CNS is a continuous process involving the generation of new myelinating mature oligodendrocytes.  Despite controversy regarding their intrinsic in vitro or in vivo lineage potential, compelling evidence indicates that a widespread proliferating adult stem cell population of nerve/glial antigen-2 (NG2)- and platelet-derived growth factor receptor alpha subunit (PDGFRα)-positive cells, termed NG2-glia or OPCs, are the major source of newly formed mature oligodendrocytes necessary for remyelination.  Studies evaluating the presence and relative densities of these OPCs at sites of chronically demyelinated MS lesions indicate that it is not a failure of numbers or migration of OPCs, but rather an inhibition of OPC differentiation at sites of injury that contributes to disease progression. 

Existing treatments for MS are based on immunosuppressive strategies that reduce relapses and delay progression.  Indeed, all approved drugs for the management of MS are powerful immunosuppressants, including corticosteroids, natalizumab, IFN-β, and FTY720, among others, some of which are associated with life-threatening side effects.  At later stages of MS, however, immunosuppressive therapies fail to influence axonal deterioration.  Thus, alternative therapies that complement immunomodulatory approaches by protecting mature oligodendrocytes and promote remyelination might be effective.

We have begun to pursue this strategy and have identified small drug-like molecules in an unbiased preliminary in vitro screen that selectively induced adult stem cell-like OPCs to differentiate into myelin basic protein (MBP)-producing mature oligodendrocytes.  This approach led to the identification of many previously identified inducers of OPC differentiation, including retinoids, thyroid hormone, corticosteroids, nucleoside analogs, Rho-kinase inhibitors, ErbB inhibitors, and ~100 hits not identified previously.  Among the ~100 confirmed hits, we identified several currently FDA-approved drugs and novel scaffolds of unknown mechanisms. 

We are further investigating these compounds' mechanistic activity in vitro and in vivo.  Importantly, several of these compounds were shown to be highly efficacious in experimental autoimmune encephalomyelitis (EAE) and the immune-independent Cuprizone model of toxic demyelination, uniquely demonstrating reductions in neurological deficits in EAE without immunosuppression.  The findings of this project have the potential to significantly impact the future development of effective treatments for MS and other demyelinating diseases.