Dual PD-1 and CTLA-4 Checkpoint Blockade Promotes Antitumor Immune Responses through CD4Foxp3 Cell-Mediated Modulation of CD103 Dendritic Cells.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy.

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy.

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs).

A Multifunctional Role for Adjuvant Anti-4-1BB Therapy in Augmenting Antitumor Response by Chimeric Antigen Receptor T Cells.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses.

A role for multiple chimeric antigen receptor-expressing leukocytes in antigen-specific responses to cancer.

While adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells can induce remission of some tumors, the role of other CAR-modified leukocytes is not well characterized. In this study, we characterize the function of leukocytes including natural killer (NK) cells, macrophages and CAR T cells from transgenic mice expressing a CAR under the control of the pan-hematopoietic promoter, vav, and determine the ability of these mice to respond to ERB expressing tumors. We demonstrate the anti-tumor functions of leukocytes, including antigen specific cytotoxicity and cytokine secretion.

Cross-talk between tumors can affect responses to therapy.

Advanced stages of cancer often involve multiple tumors in different locations in the body. These tumors are associated with a microenvironment that can influence tumor responses to immunotherapy. Whether tumors and their disparate microenvironment can interact together at distance in a multiple tumor setting, through a form of cross-talk, and affect their responses to immunotherapy has never been described.

Differential potency of regulatory T cell-mediated immunosuppression in kidney tumors compared to subcutaneous tumors.

In many cancers, regulatory T cells (Treg) play a crucial role in suppressing the effector immune response thereby permitting tumor development. Indeed, in mouse models, their depletion can promote the regression of tumors of various origins, including renal cell carcinoma when located subcutaneous (SC). In the present study, we aimed to assess the importance of Treg immunosuppression in the physiologic context of metastatic renal carcinoma (Renca) disease.

Adenosine Receptor 2A Blockade Increases the Efficacy of Anti-PD-1 through Enhanced Antitumor T-cell Responses.

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti-PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells.

Foxp3 expression in macrophages associated with RENCA tumors in mice.

The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings.

Genetic modification of mouse effector and helper T lymphocytes expressing a chimeric antigen receptor.

Genetic modification of primary mouse T cells with chimeric antigen receptors (CAR) has emerged as an important tool for optimizing adoptive T cell immunotherapy strategies. However, limitations in current protocols for generating highly pure and sufficient numbers of enriched effector and helper CAR(+) T cell subsets remain problematic. Here, we describe a new retroviral transduction protocol for successfully generating transduced CD8(+) and CD4(+) T lymphocytes for in vitro and in vivo characterization.

Blockade of PD-1 immunosuppression boosts CAR T-cell therapy.

The presence of an immunosuppressive microenvironment can limit the full potential of adoptive T cell immunotherapy. However, specific blockade of the PD-1 immunosuppressive pathway can significantly enhance the function of gene-modified T cells expressing a chimeric antigen receptor (CAR) leading to enhanced tumor eradication.

Enhancing immunotherapy using chemotherapy and radiation to modify the tumor microenvironment.

The tumor microenvironment is a complex assortment of cells that includes a variety of leukocytes. The overall effect of the microenvironment is to support the growth of tumors and suppress immune responses. Immunotherapy is a highly promising form of cancer treatment, but its efficacy can be severely compromised by an immunosuppressive tumor microenvironment.

Immune modulation of the tumor microenvironment for enhancing cancer immunotherapy.

There is much promise in the use of immunotherapy for the treatment of cancer. Approaches such as those using antibodies or adoptive cell transfer can mediate complete tumor regression in a proportion of patients. However, the tumor microenvironment can inhibit immune responses leading to ineffective or suboptimal responses of tumors to immunotherapy in the majority of cases.

Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy.

The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously.

Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors.

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A2A/A2B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.

Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells.

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression.

Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2(+) tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach.

Chimeric antigen receptor-redirected T cells display multifunctional capacity and enhanced tumor-specific cytokine secretion upon secondary ligation of chimeric receptor.

Aim: The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR).

Material & Methods: In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor.

Results: Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation.

Engineering T cell function using chimeric antigen receptors identified using a DNA library approach.

Genetic engineering of cellular function holds much promise for the treatment of a variety of diseases including gene deficiencies and cancer. However, engineering the full complement of cellular functions can be a daunting genetic exercise since many molecular triggers need to be activated to achieve complete function. In the case of T cells, genes encoding chimeric antigen receptors (CARs) covalently linking antibodies to cytoplasmic signaling domains can trigger some, but not all, cellular functions against cancer cells.

Pegasus, the 'atypical' Ikaros family member, influences left-right asymmetry and regulates pitx2 expression.

Members of the Ikaros family of zinc-finger transcription factors have been shown to be critical for immune and blood cell development. However, the role of the most divergent family member, Pegasus, has remained elusive, although it shows conservation to invertebrate Hunchback proteins that influence embryonic patterning through regulation of homeodomain genes. Zebrafish was employed as a relevant model to investigate the function of Pegasus since it possesses a single pegasus orthologue with high homology to its mammalian counterparts.

Virotherapy, gene transfer and immunostimulatory monoclonal antibodies.

Malignant cells are susceptible to viral infection and consequent cell death. Virus-induced cell death is endowed with features that are known to stimulate innate and adaptive immune responses. Thus danger signals emitted by cells succumbing to viral infection as well as viral nucleic acids are detected by specific receptors, and tumor cell antigens can be routed to professional antigen-presenting cells.

Oncolytic virus and anti-4-1BB combination therapy elicits strong antitumor immunity against established cancer.

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137).

The Ikaros gene family: transcriptional regulators of hematopoiesis and immunity.

The Ikaros family of proteins - comprising Ikaros, Aiolos, Helios, Eos and Pegasus - are zinc finger transcription factors. These proteins participate in a complex network of interactions with gene regulatory elements, other family members and a raft of other transcriptional regulators to control gene expression including via chromatin remodelling. In this way, Ikaros family members regulate important cell-fate decisions during hematopoiesis, particularly in the development of the adaptive immune system.

Origins of adaptive immunity.

Adaptive immunity, involving distinctive antibody- and cell-mediated responses to specific antigens based on "memory" of previous exposure, is a hallmark of higher vertebrates. It has been argued that adaptive immunity arose rapidly, as articulated in the "big bang theory" surrounding its origins, which stresses the importance of coincident whole-genome duplications. Through a close examination of the key molecules and molecular processes underpinning adaptive immunity, this review suggests a less-extreme model, in which adaptive immunity emerged as part of longer evolutionary journey.

Adoptive immunotherapy combined with intratumoral TLR agonist delivery eradicates established melanoma in mice.

Toll-like receptor (TLR) agonists can trigger broad inflammatory responses that elicit rapid innate immunity and promote the activities of lymphocytes, which can potentially enhance adoptive immunotherapy in the tumor-bearing setting. In the present study, we found that Polyinosinic:Polycytidylic Acid [Poly(I:C)] and CpG oligodeoxynucleotide 1826 [CpG], agonists for TLR 3 and 9, respectively, potently activated adoptively transferred T cells against a murine model of established melanoma. Intratumoral injection of Poly(I:C) and CpG, combined with systemic transfer of activated pmel-1 T cells, specific for gp100(25-33), led to enhanced survival and eradication of 9-day established subcutaneous B16F10 melanomas in a proportion of mice.

Evolution of the Ikaros gene family: implications for the origins of adaptive immunity.

Members of the Ikaros family of transcription factors are important for immune system development. Analysis of Ikaros-related genes from a range of species suggests the Ikaros family derived from a primordial gene, possibly related to the present-day protostome Hunchback genes. This duplicated before the divergence of urochordates to produce two distinct lineages: one that generated the Ikaros factor-like (IFL) 2 genes of urochordates/lower vertebrates and the Pegasus genes of higher vertebrates, and one that generated the IFL1 genes of urochordates/lower vertebrates, the IKFL1 and IKFL2 genes of agnathans and the remaining four Ikaros members of higher vertebrates.