Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers.

In order to stimulate antigen presentation and T cell activity against cancer, we treated three different tumor models in mice with the monoclonal antibodies anti-CD40 plus anti-CD137 (BiMab). In a subcutaneous transplantable MC38 colon cancer model, there was significant enhancement in the survival of mice following BiMab treatment. Anti-CD40 has shown considerable success against lymphoma in previous studies by other investigators, and we also showed in this study that, in a model of Eµ-Myc lymphoma, there was a statistically significant enhancement of survival of mice following BiMab treatment. Following the success of the BiMab treatment in the previous two models, we wished to determine if it would be successful in a mouse model of multiple myeloma. Firstly, we tested a transplantable model of disease in which multiple myeloma cells derived from Vk*MYC mice were injected intravenously. A minor proportion of anti-CD137 and BiMab treated mice experienced prolongation of life beyond 250 days. Then we tested the therapy in a spontaneously occurring multiple myeloma model, in Vk*MYC transgenic mice. The majority of mice treated survived longer than control mice, although statistical significance was not demonstrated.

PET imaging to non-invasively study immune activation leading to antitumor responses with a 4-1BB agonistic antibody.

BACKGROUND: Molecular imaging with positron emission tomography (PET) may allow the non-invasive study of the pharmacodynamic effects of agonistic monoclonal antibodies (mAb) to 4-1BB (CD137). 4-1BB is a member of the tumor necrosis factor family expressed on activated T cells and other immune cells, and activating 4-1BB antibodies are being tested for the treatment of patients with advanced cancers. METHODS: We studied the antitumor activity of 4-1BB mAb therapy using [(18) F]-labeled fluoro-2-deoxy-2-D-glucose ([(18) F]FDG) microPET scanning in a mouse model of colon cancer. Results of microPET imaging were correlated with morphological changes in tumors, draining lymph nodes as well as cell subset uptake of the metabolic PET tracer in vitro. RESULTS: The administration of 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival; complete tumor shrinkage was achieved in the majority of mice. There was markedly increased [(18) F]FDG signal at the tumor site and draining lymph nodes. In a metabolic probe in vitro uptake assay, there was an 8-fold increase in uptake of [(3)H]DDG in leukocytes extracted from tumors and draining lymph nodes of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data. CONCLUSION: Increased uptake of [(18) F]FDG by PET scans visualizes 4-1BB agonistic antibody-induced antitumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic.

Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies.

It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(α)-CD137, α-CD40] or relieve immunosuppression [α-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of α-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when α-CD137 and α-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-γ ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity.

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans.

Immunosuppressive tumor microenvironments can restrain antitumor immunity, particularly in pancreatic ductal adenocarcinoma (PDA). Because CD40 activation can reverse immune suppression and drive antitumor T cell responses, we tested the combination of an agonist CD40 antibody with gemcitabine chemotherapy in a small cohort of patients with surgically incurable PDA and observed tumor regressions in some patients. We reproduced this treatment effect in a genetically engineered mouse model of PDA and found unexpectedly that tumor regression required macrophages but not T cells or gemcitabine. CD40-activated macrophages rapidly infiltrated tumors, became tumoricidal, and facilitated the depletion of tumor stroma. Thus, cancer immune surveillance does not necessarily depend on therapy-induced T cells; rather, our findings demonstrate a CD40-dependent mechanism for targeting tumor stroma in the treatment of cancer.

Altered development of CD8+ T cell lineages in mice deficient for the Tec kinases Itk and Rlk.

Mutations affecting the Tec kinases Itk and Rlk decrease T cell receptor-induced Ca(2+) mobilization and Erk kinase activation and impair both positive and negative thymic selection. Itk(-/-) and Rlk(-/-)Itk(-/-) mice also have decreased CD4:8 T cell ratios, suggestive of altered CD4:8 lineage commitment. Nonetheless, we find that CD8 single-positive (SP) thymocytes and peripheral CD8(+) T cells in these mice do not resemble conventional CD8(+) T cells. Instead, these cells express memory markers, rapidly produce interferon-gamma, and can be selected on hematopoietically derived cells, similar to MHC class Ib-restricted "innate-type" lymphocytes. Itk deficiency also greatly increases the number of cells selected by MHC class Ib. Expression of a hypersensitive Erk2 mutant partially corrects the CD8(+) T cell phenotypes in Itk(-/-) mice, arguing that altered signaling permits development of this innate-type CD8(+) cell population. Our results suggest that Tec kinases differentially regulate development of conventional versus nonconventional lymphocytes.

Active Ca2+/calmodulin-dependent protein kinase II gamma B impairs positive selection of T cells by modulating TCR signaling.

T cell development is regulated at two critical checkpoints that involve signaling events through the TCR. These signals are propagated by kinases of the Src and Syk families, which activate several adaptor molecules to trigger Ca(2+) release and, in turn, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. In this study, we show that a constitutively active form of CaMKII antagonizes TCR signaling and impairs positive selection of thymocytes in mice. Following TCR engagement, active CaMKII decreases TCR-mediated CD3zeta chain phosphorylation and ZAP70 recruitment, preventing further downstream events. Therefore, we propose that CaMKII belongs to a negative-feedback loop that modulates the strength of the TCR signal through the tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP-2).

Dendritic epidermal T-cell activation.

Although gammadelta T cells compose a small proportion of lymphocytes in lymphoid compartments and peripheral blood, they are the major T-cell population present in epithelial tissues. However, the role played by gammadelta TCR expressing intraepithelial lymphocytes (IEL) has been enigmatic. The location of tissue-resident IEL suggests that they are important members of the first line of defense against insult for organs exposed to the environment, including the skin, gut, lungs, and reproductive system. Dendritic epidermal T cells (DETC) are the skin-resident gammadeltaIEL and serve as a model system for gammadeltaIEL in other locations. DETC have demonstrated importance in the modulation of immune responses, surveillance and repair of tissue, and resistance to infection. This work discusses recent developments in understanding DETC activation.

Gammadelta T cell-induced hyaluronan production by epithelial cells regulates inflammation.

Nonhealing wounds are a major complication of diseases such as diabetes and rheumatoid arthritis. For efficient tissue repair, inflammatory cells must infiltrate into the damaged tissue to orchestrate wound closure. Hyaluronan is involved in the inflammation associated with wound repair and binds the surface of leukocytes infiltrating damaged sites. Skin gammadelta T cells play specialized roles in keratinocyte proliferation during wound repair. Here, we show that gammadelta T cells are required for hyaluronan deposition in the extracellular matrix (ECM) and subsequent macrophage infiltration into wound sites. We describe a novel mechanism of control in which gammadelta T cell-derived keratinocyte growth factors induce epithelial cell production of hyaluronan. In turn, hyaluronan recruits macrophages to the site of damage. These results demonstrate a novel function for skin gammadelta T cells in inflammation and provide a new perspective on T cell regulation of ECM molecules.

Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1.

A fine balance between rates of proliferation and apoptosis in the skin provides a defensive barrier and a mechanism for tissue repair after damage. Vgamma3(+) dendritic epidermal T cells (DETCs) are primary modulators of skin immune responses. Here we show that DETCs both produce and respond to insulin-like growth factor 1 (IGF-1) after T cell receptor stimulation. Mice deficient in DETCs had a notable increase in epidermal apoptosis that was abrogated by the addition of DETCs or IGF-1. Furthermore, DETC-deficient mice had reduced IGF-1 receptor activation at wound sites. These findings indicate critical functions for DETC-mediated IGF-1 production in regulating skin homeostasis and repair.

Regulation of skin cell homeostasis by gamma delta T cells.

Although innate T lymphocytes such as gamma delta T cells have been extensively studied, their biological role has remained an enigma to researchers for many years. However, recent advances have begun to explain their complex role in the immune system. Gamma delta T cells are often the major T cell population in epithelial tissues such as the skin, gut, and lung where they have been implicated in maintaining tissue integrity, defending against pathogens, and regulating inflammation. The gamma delta T cells that reside in the skin are a prototypical intra-epithelial lymphocyte (IEL) population. These skin gamma delta T cell receptor (TCR)-expressing cells are named dendritic epidermal T cells (DETC) for their unique dendritic morphology. Using their gamma delta TCR, DETC recognize an unknown ligand expressed by stressed or damaged keratinocytes. Activated DETC exhibit effector functions such as cytokine production, cytolysis, and proliferation in vitro. Recent findings have shown that upon activation by damaged keratinocytes, DETC produce a key keratinocyte growth factor for wound repair called fibroblast growth factor 7 (FGF-7). FGF-7 is produced in vitro and in vivo, suggesting that DETC might play an important role in the biological function of wound repair. Indeed a delay in wound closure and a decrease in the proliferation of keratinocytes at the wound site have been observed in mice lacking gamma delta T cells. In addition to effector functions attributed to DETC, it has also been suggested that gamma delta T cells such as DETC have regulatory roles such as initiating or inhibiting inflammation. This is supported by the findings that DETC produce chemokines and cytokines. Control of the inflammatory response in the epithelium may provide another mechanism to reestablish homeostasis after a biological insult such as wound infliction. Understanding the function of DETC may be useful in the development of future therapies for chronic wounds and the maintenance of skin homeostasis.

Preferential activation of an IL-2 regulatory sequence transgene in TCR gamma delta and NKT cells: subset-specific differences in IL-2 regulation.

A transgene with 8.4-kb of regulatory sequence from the murine IL-2 gene drives consistent expression of a green fluorescent protein (GFP) reporter gene in all cell types that normally express IL-2. However, quantitative analysis of this expression shows that different T cell subsets within the same mouse show divergent abilities to express the transgene as compared with endogenous IL-2 genes. TCR gamma delta cells, as well as alpha beta TCR-NKT cells, exhibit higher in vivo transgene expression levels than TCR alpha beta cells. This deviates from patterns of normal IL-2 expression and from expression of an IL-2-GFP knock-in. Peripheral TCR gamma delta cells accumulate GFP RNA faster than endogenous IL-2 RNA upon stimulation, whereas TCR alpha beta cells express more IL-2 than GFP RNA. In TCR gamma delta cells, IL-2-producing cells are a subset of the GFP-expressing cells, whereas in TCR alpha beta cells, endogenous IL-2 is more likely to be expressed without GFP. These results are seen in multiple independent transgenic lines and thus reflect functional properties of the transgene sequences, rather than copy number or integration site effects. The high ratio of GFP: endogenous IL-2 gene expression in transgenic TCR gamma delta cells may be explained by subset-specific IL-2 gene regulatory elements mapping outside of the 8.4-kb transgene regulatory sequence, as well as accelerated kinetics of endogenous IL-2 RNA degradation in TCR gamma delta cells. The high levels and percentages of transgene expression in thymic and splenic TCR gamma delta and NKT cells, as well as skin TCR gamma delta-dendritic epidermal T cells, indicate that the IL-2-GFP-transgenic mice may provide valuable tracers for detecting developmental and activation events in these lineages.