American Association for the Advancement of Science
For his research accomplishments, Innate Immunity Co-Leader John Byrd, MD, has been named a Fellow of the AAAS.
University Distinguished Scholar
Innate Immunity Co-Leader John Byrd, MD, was named The Ohio State University’s 2008 University Distinguished Scholar.
Natural Killer Cell Development & Biology
Innate Immunity member, OSUCCC Director and James Cancer Hospital CEO Michael A. Caligiuri, MD, and his team are studying Natural Killer cell origins and effector functions in order to develop new treatments for cancer. As a result, the group is widely recognized for their influence on the innate immunity field.
Additionally, two research projects (one analyzing signals that shape NK cell development and maturation, the other focusing on signaling pathways induced by monocyte-derived factors and their effects on NK cell cytotoxic activity and cytokine production) have translational components that resulted in novel clinical trials for patients with hematologic malignancies and solid tumors.
Natural Killer Cell Development in Lymphoid Tissues
Innate Immunity member, OSUCCC Director and James Cancer Hospital CEO Michael A. Caligiuri, MD, and his team have identified a novel CD34dimCD45RA+ hematopoietic precursor cell (HPC) that expresses high levels of the integrin alpha4beta7 (Immunity, 2005). This subset of HPCs constitutes <1% of bone marrow CD34+ HPCs and approximately 6% of blood CD34+ HPCs but >95% of lymph node CD34+ HPCs. When stimulated by IL-15 or activated lymph node T cells, these cells develop into CD56bright NK cells. These data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in lymph nodes where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.
Also, a secondary lymphoid tissues analysis identified four novel populations that differed in their expression of CD56, CD34, CD177 and CD94 and appeared to represent discrete stages of a human NK cell developmental continuum (J. Exp. Med. 2006).
Natural Killer Cell Signal Transduction & Activation in Response to Monokines
NK cell researchers have pioneered the investigation of interactions between NK cells and macrophages in response to microbial challenges and following exposure to transformed cells.
The group has shown that macrophage activation leads to release of monokines such as IL-12, IL 15 and IL-18, which induce a robust program of cytokine production by NK cells. NK cell- derived factors, such as IFN‑γ, in turn provide macrophages and T cells with signals that are important for their further activation.
TGF-ß Regulation of IFN‑γ Production
NK cell researchers have shown that T-BET-driven IFN‑γ production by human NK cells is negatively regulated by anti-inflammatory TGF-ß-induced signaling intermediates SMAD2, SMAD3 and SMAD4, whereas pro-inflammatory monokines IL-12, IL-15 and IL-18 antagonize TGF-ß signaling by down-regulating expression of the TGF-ß type II receptor and its signaling intermediates SMAD2 and SMAD3 (Immunity, 2006).
Importantly, NK cells from SMAD3-/- mice produce more IFN‑γ in vivo than NK cells from wild-type mice. TGF-ß also inhibited NK cell production of IFN‑γ in response to Ab-coated tumor targets, an effect that was mediated via SMAD3 and inhibition of T-BET. TGF-ß treatments also inhibited NK cell ADCC of Ab-coated tumor targets via a SMAD-3-dependent inhibition of granzyme A and B (J. Immunol. 2008).
Hlx and Sp-1 Regulate IFN‑γ in NK Cells
Dr. Benson and his collaborators have discovered six Sp1-binding sites within the proximal human T-BET promoter that are highly conserved among mammalian species. Electrophoretic mobility shift assays demonstrated a physical association between Sp1 and the proximal T-BET promoter, the activity of which correlated precisely with expression levels of Sp-1.
Ectopic over-expression of Sp1 also enhanced T-BET expression and cytokine-induced IFN‑γ secretion in NK cells and T cells. Mithramycin A, which blocks the binding of Sp1 to the T-BET promoter, diminished both T-BET expression and IFN‑γ production in monokine-stimulated primary human NK cells. These results suggest that Sp1 is a positive transcriptional regulator of T-BET and that targeting Sp1 may be a useful means for preventing and/or treating diseases associated with aberrant T-BET or IFN‑γ expression. (Eur. J. Immunol, 2007).
Synergy Between IL-12 & Trastuzumab
Innate Immunity Co-Leader William Carson III, MD, and his team determined that co-administration of trastuzumab and IL-12 can activate a potent cytokine secretion program within NK cells that has anti-tumor activity in vivo. Using a novel in vitro co-culture system in which trastuzumab-coated human breast cancer cells are cultured with purified human NK cells in the presence of IL-12, Dr. Carson’s group showed that trastuzumab and IL 12 synergize to induce potent cytokine secretion. The combination of IL-12 with trastuzumab-coated tumor stimulated NK cells to secrete 15-fold greater amounts of IFN‑γ protein as compared to stimulation with either agent alone – a result that could not be duplicated by co-stimulation with IL-2, IL-10, IL-15 or IL-18.
Combination Therapy with mAbs & Cytokines
An NCI-sponsored Phase I trial of IL-12 and trastuzumab for patients with HER2 (+) cancers was conducted under the auspices of the U01 program in Experimental Therapeutics (Clin. Cancer Res. 2004).
Following a loading dose, trastuzumab was administered weekly at 2 mg/kg (d1). IL-12 was given intravenously on days 2 and 5 of the weekly cycle and was dose-escalated within cohorts of three patients. The regimen was well-tolerated, and of 15 patients, there was one complete response in a patient with HER2 3+ breast cancer metastatic to loco-regional lymph nodes, and two other patients had prolonged stabilization of disease.
Correlative assays showed sustained production of IFN‑γ by NK cells only in those patients exhibiting a favorable outcome in response to the regimen. Progression-free survival also correlated with the presence of circulating IFN‑γ (nonparametric Mann-Whitney U test, P< 0.004). However, the ability of patient peripheral blood cells to conduct ADCC against tumor targets in vitro did not correlate with clinical response or dose of IL-12.
, and IP 10 and MIG were also elevated over the course of multiple cycles in patients exhibiting a clinical benefit from treatment.
Use of Cytokines with mAbs
Innate Immunity Co-Leader William Carson III, MD, and Innate Immunity member Susheela Tridandapani, PhD, and her team have shown that NK cells stimulated with antibody-coated tumor cells and IL-2 or IL-12 secrete an array of T cell attracting chemokines, including MIP-1a, MCP-1, RANTES, IL-8 and MDC (Cancer Res. 2006). These chemokines exerted potent T cell recruiting activity, as NK cell-derived culture supernatants induced significant migration of both naïve and activated T cells in an in vitro chemotaxis assay.
In addition, mice receiving IL-12 and HER2+ tumor cells coated with trastuzumab exhibited enhanced systemic levels of the chemokine MIP-1a as compared with mice receiving either agent alone. As a clinical correlate, increased circulating levels of NK cell-derived chemokines were detected within the sera of patients who exhibited a clinical response or significant disease stabilization during therapy with trastuzumab and IL-12. Sera from these patients were able to direct T-cell migration in a chemotaxis assay, and individual chemokine neutralization abrogated this effect.
These data are the first to show chemokine production by NK cells in response to stimulation with Ab-coated tumor cells, and they suggest a potential role for NK cell-derived chemokines in patients receiving therapeutic mAbs. This group's further studies determined that NK cell activating cytokines such as IL-2, IL-12 or IL-21 can similarly potentiate the innate immune response to EGFR (HER1) positive targets reated with cetuximab (Clin. Cancer. Res. 2007).
NK Cells Respond Directly to TLR Agonist
To stimulate innate immune responses for disease treatment, Innate Immunity investigators developed synthetic oligodeoxynucleotides (ODN) containing a high frequency of CpG dinucleotides. These CpG ODN, like intact bacterial DNA, act as agonists for TLR9, one of the key receptors for bacterial components.
Innate Immunity has initiated a clinical trial of a TLR9 agonist in combination with trastuzumab in patients with metastatic HER2+ breast cancer who have stabilized on trastuzumab regimens that employ cytotoxic chemotherapy.
TLR7 Agonists Act on Innate Immune Cells
Pre-clinical studies by Innate Immunity Co-Leader William Carson III, MD, and his group have shown that TLR7 agonists can enhance NK cell activity, and Innate Immunity member Susheela Tridandapani’s group has shown that they down-regulate inhibitory FcR on monocytes.
BecauseTLR7 agonist Imiquimod (Aldara) has anti-cancer activity when applied topically to superficial basal cell carcinomas, the Innate Immunity investigators determined that TLR7 agonists may have activity when administered topically or intra-tumorally to patients with locally advanced melanoma.
Regulation Macrophage FcR Expression and Signal Transduction
Innate Immunity member Susheela Tridandapani, PhD, and her team have established that the phagocytic process is subject to multiple regulatory mechanisms. One level of regulation occurs by the modulation of the ratio of activating to inhibiting FcγR on monocytes and macrophages. (Molecular Immunology, 2006)
The investigators determined that cytokines such as transforming growth factor beta (TGF-b), which have profound inhibitory effects on multiple immune compartments, exert negative influences on macrophages largely via down-regulation of stimulatory FcR.
A second level of regulation is mediated by intracellular phosphatases such as SHIP, an inositol phosphatase. Innate Immunity investigators found that SHIP-2 negatively regulates macrophage production of superoxide anion and the pro-inflammatory cytokines, TNF-a,
and IL-6 following exposure of these cells to Ab-coated targets and TLR agonists such as LPS.
Macrophage Inflammatory Cytokines
Innate Immunity member Mark Wewers, MD, and his group have demonstrated that neutrophils and endothelial cells release a potent antimicrobial peptide known as LL37 that initiates the maturation and release of IL-1ß from monocytes via its interaction with the P2X(7) receptor (J. Immunol. 2004).
Biology of FcRn
Innate Immunity member Clark Anderson, MD, has shown that FcRn (neonatal Fc receptor) works throughout the adult body to transport nonspecifically endocytosed IgG molecules to the cell exterior so that they can continue to circulate in the tissues, thus avoiding a degradative lysosomal fate. Dr. Anderson’s research provides direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis.
FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease and improving drug efficacy. A landmark paper from Dr. Anderson’s group (J. Exp. Med. 2003 and Clin Immunol. 2007) demonstrates that FcRn also rescues albumin from intracellular catabolic degradation, as it does for IgG, thus accounting for the uniquely long half-lives of both molecules.
Innate Immunity continues to increase the translation of basic immunologic research into clinical practice. In the past few years, this increase has prompted more than 30 investigator-initiated clinical trials, and more than 300 patients have been accrued to therapeutic clinical trials.
Basic Research Leading to New Clinical Trials
Innate Immunity researchers working with the Experimental Therapeutics (ET) research program have generated three successful clinical trials linking the Innate Immunity interests in interferon biology with ET efforts.
Administered through the U01 mechanism and funded by an NCI Quick Trial grant, each trial rapidly met its full accrual goals:
- A Phase I Study of Sequential Vaccinations with Fowlpox-CEA (6D)-TRICOM and Vaccinia-CEA (6D)-TRICOM, in Combination with GM-CSF and IFN-a-2B in Patients with CEA Expressing Carcinomas. NCI 5633
- A Phase 2 Study of Bevacizumab and High-Dose Interferon-alpha-2b in Metastatic Malignant Melanoma. NCI 2669
- A Phase I Study of PS-341 (Bortezomib, Velcade) and Interferon-alpha-2b in Malignant Melanoma.
Melanoma and Renal Cell Carcinoma Therapy
Phase I Trial of IL-12 plus IFN-a
Innate Immunity investigators were the first investigator to show that pre-treating cancer cells with IFN-γ led to marked upregulation of the Jak-STAT proteins involved in interferon receptor signal transduction. He subsequently demonstrated that IL-12 could induce the endogenous production of IFN-γ in mice bearing melanoma tumors and that this pre-treatment could markedly enhance the activity of subsequent low-dose IFN-a therapy (J. Immunol. 2004). Title: Phase I Study of the Sequential Combination of Interleukin-12 and Interferon Alfa-2b in Advanced Cancer. P.I. W. Carson. Sponsor: NCI, CTEP. Funding: NCI R21 CA84402 (correlative studies).
Phase II Trial of IL-12 plus IFN-a
Innate Immunity Co-Leader William Carson III, MD, successfully translated Phase I data into a national trial of IL-12 and IFN-a in patients with stage IV melanoma within the CALGB cooperative group.
The trial concluded successfully last year when the study’s last patient progressed after experiencing a partial response and 34 months of disease stabilization. This regimen was well-tolerated and a modest level of clinical activity was observed: two patients with partial responses and six with stable disease out of a total of 36 patients. Correlative studies revealed that IL-12 was a potent inducer of IFN‑γ and that levels of this cytokine correlated with the induction of STAT1 and STAT2 levels in patient PBMCs, which in turn augmented the ability of low-dose IFN-a to stimulate the formation of phospho-STAT1 (activated form). The findings will be published and will form the foundation for future trials of cytokine therapy in melanoma at OSU. Title: CALGB 500001: Phase II Trial of Interleukin-12 Followed by Interferon Alfa-2B in Patients with Metastatic Malignant Melanoma. PI W. Carson. Sponsor: CALGB. Funding: NCI R21 CA92286 (correlative studies).
Bortezomib plus IFN‑α
Innate Immunity Co-Leader William Carson III, MD, and his group have shown that inhibition of the proteasome in melanoma cells (e.g., with bortezomib) synergistically enhances the apoptotic effects of IFN‑α . Careful in vitro studies suggested that bortezomib and IFN-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Bortezomib and IFN‑α displayed statistically significant anti-tumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts (Cancer Res. 2008 and Cancer Imm. Imm. 2009). These data supported an investigator-initiated, pharmaceutical-sponsored trial of bortezomib and IFN‑α for patients with metastatic melanoma that finished accruing in August, 2009.
Personalized IFN‑α Therapy
Innate Immunity investigators Greg Lesinski and Kari Kendra ahve investigated melanoma cell response to IFN‑α and found that IFN‑α induced significantly lower levels of activated (i.e. phosphorylated) [P]-STAT1 in patient melanoma cells compared with matched PBMCs (Clin. Cancer. Res. 2007). Furthermore, STAT1 and STAT2 expression in malignant melanoma cells did not correlate with response to IFN-a adjuvant therapy (Cancer Imm. Imm. 2005).
By examining this signaling pathway’s role in a murine model of malignant melanoma, the investigators discovered that STAT1 signal transduction within the host immune system (and not the tumor cell) was responsible for mediating the anti-tumor effects of IFN‑α (J. Clin. Investig. 2003 and J. Surg Res. 2004).
The group went on to develop an intracellular flow cytometric technique for analyzing STAT1 activation within specific immune cell subsets using an antibody that recognizes the tyrosine phosphorylated form of STAT1 (JNCI, 2004). This technique represents an exciting new way to analyze the activation state of immune cells during the course of cytokine therapy because it is rapid, highly quantitative and uses fewer cells than other assays.
Cancer Therapy Vaccine
Innate Immunity member, OSUCCC Director and James Cancer Hospital CEO Michael A. Caligiuri, MD, has spearheaded what could be the first trial assessing reduced immune suppression effects in conjunction with anti-viral drug therapy for patients with a form of lymphoma known as post-transplant lymphoproliferative disorder, or PTLD (Blood, 2002 and 2005). This intervention’s success led his group to begin developing an EBV vaccine based on the TRICOM backbone that would be given to patients prior to organ transplantation in an effort to prevent the onset of PTLD.
The Innate Immunity research program is one of OSUCCC–James' six highly interactive cancer Research Programs. The programs comprise more than 200 leading scientists from 13 different colleges within Ohio State and affiliated academic institutions.
Innate Immunity investigators collaborate with several OSUCCC–James Shared Resources, including the Clinical Trials shared resource, which is integral to the success of many of the Innate Immunity’s new endeavors as well as the Biostatistics shared resource –another heavily utilized SR in many funded studies, the Microscopy Shared Resource, the Nucleic Acid shared resource, the Proteomics Shared Resource, the Microscopy shared resource, the Cytometry shared resource, the Biorepository and Biospecimen shared resource and the Comparative Pathology and Mouse Phenotyping Shared Resource.
Other collaborations include:
Intra- and Inter-Programmatic Collaboration
The ET SPORE Grant in leukemia includes investigators from Innate Immunity, ET and the Molecular Biology and Cancer Genetics research program. Project 3, led by Innate Immunity Co-Leader John Byrd, MD, focuses on the mechanism of immune activation of the immunomodulatory agent lenalidomide and strategies to enhance its activity with other biologic therapies. Other projects in the SPORE focus on the molecular genetics of leukemia and novel therapeutics for CLL.
Innate Immunity Co-Leader William Carson III, PhD, has collaborated with OSU Department of Biomedical Engineering’s James Lee, PhD, to develop nanodevices that can release immunotherapeutic agents at a constant rate into the tumor microenvironment. These devices comprise biodegradable micro-reservoirs fitted with nanochannel membranes that regulate biologically active proteins’ constant and ordered release.
Innate Immunity member Clay Marsh, MD, and other Innate Immunity investigators are collaborating with ET researchers to characterize miRNA patterns in specific types of cancer before and after Phase I therapy. The groups had previously collaborated on a study of circulating microRNAs with immune modulatory effects.
A collaboration between Innate Immunity and MBCG investigators demonstrated that culture of CD34+ HPCs or primary human NK cells in IL-2 or IL-15 and c-Kit ligand (KL) resulted in enhanced growth compared with either cytokine alone. NK cell activation by IL 2/15 increased the expression of c-kit, and this receptor’s kinase activity was required for synergy between KL and IL 2/15. The synergistic proliferation was dependent on mitogen-activated protein kinase (MAPK) signaling, and the effects were mediated at least in part by modulation of CDK4 and p27 expression that permitted earlier entry of NK cells into the cell cycle. This long-standing area of investigation (14 publications) led directly to a Phase I study of ultra-low dose IL-2 and c-Kit ligand in patients with HIV infection and cancer.
Given the widespread use of trastuzumab with cytotoxic agents, Innate Immunity and the ET investigators collaborated on a clinical trial of IL-12 in combination with trastuzumab plus paclitaxel.
Innate Immunity and Cancer Control research program (CCRP) investigators collaborated on a study showing that SHIP-2 negatively regulates macrophage production of superoxide anion and the pro-inflammatory cytokines,
and IL-6, after exposing the cells to Ab-coated targets and TLR agonists such as LPS.
In order to further identify proteins with the potential to bind to key phosphatases (such as SHIP) and thereby modulate their activity, Innate Immunity member Susheela Tridandapani, PhD, has collaborated with the OSU Department of Chemistry’s Dehua Pei, PhD, a Molecular Carcinogenesis and Chemoprevention Research Program member.
A collaboration between Innate Immunity P01 researchers and ET researchers determined that the NK cell response to HER1+ squamous carcinoma cell lines is markedly enhanced by cytokines such as IL-12. (Title: Phase I Trial of Interleukin-12 in Combination With Cetuximab in Patients With HER1-Positive Oral Cancers. PI: Ted Teknos. Sponsor: NCI, CTEP. )
Innate Immunity investigators and ET member Tanios Bekaii-Saab, MD, are collaborating to develop a clinical trial based on P01 researchers’ pre-clinical studies, which determined that IL-21 is a potent adjuvant to cetuximab therapy in pancreatic cancer patients. (Title: Phase I Trial of Interleukin-21 in Combination With Cetuximab in Patients With Metastatic Pancreatic Cancer. PI: Tanios Bekaii-Saab. Sponsor: NCI, CTEP.)
Cancer Control research program members Barbara Andersen, PhD, Ronald Glaser, PhD, and Janice Kiecolt-Glaser, PhD, have collaborated with Innate Immunity Co-Leader William Carson III., PhD, to explore the effects of stress on breast cancer patients’ immune systems.