Oncolytic Adenovirus Armed with a Novel Agonist of the CD137 Immune Checkpoint Stimulator Suppresses Tumor Growth.

Natural 4-1BBL (CD137L) is a cell membrane-bound protein critical to the expansion, effector function, and survival of CD8+ T cells. We reported the generation of an active soluble oligomeric construct, SA-4-1BBL, with demonstrated immunoprevention and immunotherapeutic efficacy in various mouse tumor models. Herein, we developed an oncolytic adenovirus (OAd) for the delivery and expression of SA-4-1BBL (OAdSA-4-1BBL) into solid tumors for immunotherapy. SA-4-1BBL protein expressed by this construct produced T-cell proliferation in vitro. OAdSA-4-1BBL decreased cell viability in two mouse lung cancer cell lines, TC-1 and CMT64, but not in the non-cancerous lung MM14.Lu cell line. OAdSA-4-1BBL induced programmed cell death types I and II (apoptosis and autophagy, respectively), and autophagy-mediated adenosine triphosphate (ATP) release was also detected. Intratumoral injection of OAdSA-4-1BBL efficiently expressed the SA-4-1BBL protein in the tumors, resulting in significant tumor suppression in a syngeneic subcutaneous TC-1 mouse lung cancer model. Tumor suppression was associated with a higher frequency of dendritic cells and an increased infiltration of cytotoxic CD8+ T and NK cells into the tumors. Our data suggest that OAdSA-4-1BBL may present an efficacious alternative therapeutic strategy against lung cancer as a standalone construct or in combination with other immunotherapeutic modalities, such as immune checkpoint inhibitors.

Protein Phosphatase 2A: Role in T Cells and Diseases.

Protein phosphatase 2A (PP2A) is a serine-threonine phosphatase that plays an important role in the regulation of cell proliferation and signal transduction. The catalytic activity of PP2A is integral in the maintenance of physiological functions which gets severely impaired in its absence. PP2A plays an essential role in the activation, differentiation, and functions of T cells. PP2A suppresses Th1 cell differentiation while promoting Th2 cell differentiation. PP2A fosters Th17 cell differentiation which contributes to the pathogenesis of systemic lupus erythematosus (SLE) by enhancing the transactivation of the Il17 gene. Genetic deletion of PP2A in Tregs disrupts Foxp3 expression due to hyperactivation of mTORC1 signaling which impairs the development and immunosuppressive functions of Tregs. PP2A is important in the induction of Th9 cells and promotes their antitumor functions. PP2A activation has shown to reduce neuroinflammation in a mouse model of experimental autoimmune encephalomyelitis (EAE) and is now used to treat multiple sclerosis (MS) clinically. In this review, we will discuss the structure and functions of PP2A in T cell differentiation and diseases and therapeutic applications of PP2A-mediated immunotherapy.

Engineering donor lymphocytes with Fas ligand protein effectively prevents acute graft-versus-host disease.

Alloreactive T-effector cells (Teffs) are the major culprit of acute graft-versus-host disease (aGVHD) associated with hematopoietic stem cell transplantation. Ex vivo nonspecific depletion of T cells from the donor graft impedes stem cell engraftment and posttransplant immune reconstitution. Teffs upregulate Fas after activation and undergo Fas ligand (FasL)-mediated restimulation-induced cell death (RICD), an important mechanism of immune homeostasis. We targeted RICD as a means to eliminate host-reactive Teffs in vivo for the prevention of aGVHD. A novel form of FasL protein chimeric with streptavidin (SA-FasL) was transiently displayed on the surface of biotinylated lymphocytes, taking advantage of the high-affinity interaction between biotin and streptavidin. SA-FasL-engineered mouse and human T cells underwent apoptosis after activation in response to alloantigens in vitro and in vivo. SA-FasL on splenocytes was effective in preventing aGVHD in >70% of lethally irradiated haploidentical mouse recipients after cotransplantation with bone marrow cells, whereas all controls that underwent transplantation with nonengineered splenocytes developed aGVHD. Prevention of aGVHD was associated with an increased ratio of CD4+CD25+FoxP3+ T regulatory (Tregs) to Teffs and significantly reduced transcripts for proinflammatory cytokines in the lymphoid organs and target tissues. Depletion of Tregs from the donor graft abrogated the protection conferred by SA-FasL. This approach was also effective in a xenogeneic aGVHD setting where SA-FasL-engineered human PBMCs were transplanted into NSG mice. Direct display of SA-FasL protein on donor cells as an effective means of eliminating alloreactive Teffs in the host represents a practical approach with significant translation potential for the prevention of aGVHD.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long-term graft-localized immune privilege.

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA-FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long-term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA-FasL-engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor-matched, but not third-party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft-draining lymph nodes, and required phagocytes and TGF-ß. At the maintenance phase, immune protection evolved into graft site-restricted immune privilege as the destruction of long-surviving SA-FasL-islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4+ CD25+ Foxp3+ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA-FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.

A Novel Form of 4-1BBL Prevents Cancer Development via Nonspecific Activation of CD4+ T and Natural Killer Cells.

Costimulation through 4-1BB (CD137) receptor generates robust CD8+ T-effector and memory responses. The only known ligand, 4-1BBL, is a trimeric transmembrane protein that has no costimulatory activity as a soluble molecule. Thus, agonistic antibodies to the receptor have been used for cancer immunotherapy in preclinical models and are currently being evaluated in the clinic. Here, we report that treatment with an oligomeric form of the ligand, SA-4-1BBL, as a single agent is able to protect mice against subsequent tumor challenge irrespective of the tumor type. Protection was long-lasting (>8 weeks) and a bona fide property of SA-4-1BBL, as treatment with an agonistic antibody to the 4-1BB receptor was ineffective in generating immune protection against tumor challenge. Mechanistically, SA-4-1BBL significantly expanded IFNγ-expressing, preexisting memory-like CD44+CD4+ T cells and NK cells in naïve mice as compared with the agonistic antibody. In vivo blockade of IFNγ or depletion of CD4+ T or NK cells, but not CD8+ T or B cells, abrogated the immunopreventive effects of SA-4-1BBL against cancer. SA-4-1BBL as a single agent also exhibited robust efficacy in controlling postsurgical recurrences. This work highlights unexpected features of SA-4-1BBL as a novel immunomodulator with implications for cancer immunoprevention and therapy. SIGNIFICANCE: This study demonstrates the unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with both preventive and therapeutic efficacy against cancer.

Current challenges for cancer vaccine adjuvant development.

INTRODUCTION: Although much progress has been made in the last decade(s) toward development of effective cancer vaccines, there are still important obstacles to therapeutic successes. New generations of cancer vaccines will benefit from a combination adjuvant approach that targets multiple branches of the immune response. AREAS COVERED: Herein we describe how combinatorial adjuvant strategies can help overcome important obstacles to cancer vaccine development, including antigen immunogenicity and tumor immune suppression. Tumor antigens may be both tolerogenic and may utilize active mechanisms to suppress host immunity, including downregulation of MHC molecules to evade recognition and upregulation of immune inhibitory receptors, to subvert an effective immune response. The current cancer vaccine literature was surveyed to identify advancements in the understanding of the biological mechanisms underlying poor antigen immunogenicity and tumor immune evasion, as well as adjuvant strategies designed to overcome them. EXPERT COMMENTARY: Poor immunogenicity of tumor antigens and tumor immune evasion mechanisms make the design of cancer vaccines challenging. Growing understanding of the tumor microenvironment and associated immune responses indicate the importance of augmenting not only the effector response, but also overcoming the endogenous regulatory response and tumor evasion mechanisms. Therefore, new vaccines will benefit from multi-adjuvanted approaches that simultaneously stimulate immunity while preventing inhibition.