Grazyna Lipowska-Bhalla

Acquired Resistance to Fractionated Radiotherapy Can Be Overcome by Concurrent PD-L1 Blockade

Radiotherapy is a major part in the treatment of most common cancers, but many patients experience local recurrence with metastatic disease. In evaluating response biomarkers, we found that low doses of fractionated radiotherapy led to PD-L1 upregulation on tumor cells in a variety of syngeneic mouse models of cancer. Notably, fractionated radiotherapy delivered in combination with αPD-1 or αPD-L1 mAbs generated efficacious CD8(+) T-cell responses that improved local tumor control, long-term survival, and protection against tumor rechallenge. These favorable outcomes were associated with induction of a tumor antigen-specific memory immune response. Mechanistic investigations showed that IFNγ produced by CD8(+) T cells was responsible for mediating PD-L1 upregulation on tumor cells after delivery of fractionated radiotherapy. Scheduling of anti-PD-L1 mAb was important for therapeutic outcome, with concomitant but not sequential administration with fractionated radiotherapy required to improve survival. Taken together, our results reveal the mechanistic basis for an adaptive response by tumor cells that mediates resistance to fractionated radiotherapy and its treatment failure. With attention to scheduling, combination immunoradiotherapy with radiotherapy and PD-1/PD-L1 signaling blockade may offer an immediate strategy for clinical evaluation to improve treatment outcomes.

Targeted immunotherapy of cancer with CAR T cells: achievements and challenges

The adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a relatively new but promising approach in the field of cancer immunotherapy. This therapeutic strategy is based on the genetic reprogramming of T cells with an artificial immune receptor that redirects them against targets on malignant cells and enables their destruction by exerting T cell effector functions. There has been an explosion of interest in the use of CAR T cells as an immunotherapy for cancer. In the pre-clinical setting, there has been a considerable focus upon optimizing the structural and signaling potency of the CAR while advances in bio-processing technology now mean that the clinical testing of these gene-modified T cells has become a reality. This review will summarize the concept of CAR-based immunotherapy and recent clinical trial activity and will further discuss some of the likely future challenges facing CAR-modified T cell therapies.

Antitumor efficacy of radiation plus immunotherapy depends upon dendritic cell activation of effector CD8+ T cells

Radiotherapy plus CD40 or TLR7 stimulation leads to long-term clearance of B- and T-cell lymphomas. These curative responses after combining radiotherapy with an immunomodulatory agent depended upon the priming of tumor-specific CD8+ T cells by dendritic cells. Tumor cells dying after cytotoxic therapy are a potential source of antigen for T-cell priming. Antigen-presenting cells (APC) can cross-present MHC I–restricted peptides after the uptake of dying cells. Depending on the nature of the surrounding environmental signals, APCs then orchestrate a spectrum of responses ranging from immune activation to inhibition. Previously, we had demonstrated that combining radiation with either agonistic monoclonal antibody (mAb) to CD40 or a systemically administered TLR7 agonist could enhance CD8 T-cell–dependent protection against syngeneic murine lymphoma models. However, it remains unknown how individual APC populations affect this antitumor immune response. Using APC depletion models, we now show that dendritic cells (DC), but not macrophages or B cells, were responsible for the generation of long-term immunologic protection following combination therapy with radiotherapy and either agonistic CD40 mAb or systemic TLR7 agonist therapy. Novel immunotherapeutic approaches that augment antigen uptake and presentation by DCs may further enhance the generation of therapeutic antitumor immune responses, leading to improved outcomes after radiotherapy. Cancer Immunol Res; 4(7); 621–30. ©2016 AACR.

A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells

Anti-CD20 monoclonal antibodies (mAb) such as rituximab have been proven to be highly effective at improving outcome in B-cell malignancies. However, many patients ultimately relapse and become refractory to treatment. The glycoengineered anti-CD20 mAb obinutuzumab was developed to induce enhanced antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis and direct cell death and was shown to lead to improved outcomes in a randomized study in B-CLL. We hypothesized that immune stimulation through Toll-like receptor 7 (TLR7) agonism in combination with obinutuzumab would further enhance lymphoma clearance and the generation of long-term antitumor immune responses. Here we demonstrate, in syngeneic human CD20 (hCD20)-expressing models of lymphoma, that systemic administration of a TLR7 agonist (R848) increases responses when administered in combination with obinutuzumab and protects against disease recurrence. Depletion studies demonstrate that primary antitumor activity is dependent on both NK cells and CD4+ T cells but not on CD8+ T cells. However, both CD4+ and CD8+ T cells appear necessary for the generation of protective immunological memory. Importantly, increased tumor-free survival post obinutuzumab and R848 combination therapy was seen in hCD20 transgenic mice, which express hCD20 on normal B cells. These findings provide a rationale for clinical testing of obinutuzumab in combination with systemically administered TLR7 agonists to further improve outcome.

The anti-tumor immune response generated by radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade.

Radiation therapy (RT) plays a definitive part of anti-cancer therapy for the majority of common cancers but for many patients, metastatic disease and local recurrence are common and the outlook remains poor. New more effective RT combination approaches are urgently required that decrease local and distant recurrence to improve outcomes. Using a range of established syngeneic tumor models (CT26 (colorectal), 4T1 (breast) or 4434 (BRafV600E p16-/- melanoma) we sought to determine the impact of fractionated RT (fRT) on the tumor microenvironment. Our data reveal that treatment with a course of fRT leads to significant upregulation of tumor cell expression of PD-L1 in vivo. Through cellular depletion studies we determined that this RT-mediated increase in tumor cell expression of PD-L1 was dependent on CD8+ T cells. Furthermore, ShRNA-mediated silencing of tumor cell IFNγR1 expression or administration of an IFNγ depleting mAb phenocopied the depletion of CD8+ T cells. Taken together, these data demonstrate that adaptive upregulation of tumor cell PD-L1 following treatment with low-dose fRT is mediated by tumor infiltrating CD8+ T cell production of IFNγ. Using a dual tumor model our data reveal that this adaptive upregulation is restricted to the irradiated tumor site with no change in tumor cell PD-L1 expression detected in tumors situated outside of the ionizing radiation field, signifying that treatment with RT alone may not generate systemic tumor antigen-specific responses. Administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with fRT was found to substantially improve survival when compared to either monotherapy alone. In addition, abscopal responses were observed on tumors outside of the RT treatment field. Our data reveal that up to 60% of mice undergo a complete response following combination therapy and are protected against tumor rechallenge by the generation of long-term immunological memory. Furthermore, we found that scheduling of anti-PD-L1 mAb relative to the delivery of fRT appeared important to therapeutic outcome with concomitant but not sequential administration required for improved survival. Tumor cell PD-L1 expression following treatment with fRT appears to be a mechanism of adaptive immunological resistance which may potentially contribute to fRT treatment failure. This study demonstrates the potential for enhancing the efficacy of conventional RT through blockade of the PD-1/PD-L1 axis, but sequencing is critical, providing important new insights for clinical evaluation.

Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.

Improving therapeutic activity of anti-CD20 antibody therapy through immunomodulation in lymphoid malignancies

Abstract Nearly two decades ago rituximab heralded a new era in management of B cell malignancies significantly increasing response rates and survival. However, despite clear therapeutic advantage, significant numbers of patients become refractory to anti-CD20 mAb therapy, suggesting urgent improvements are required. It is now well recognized that the suppressive tumor microenvironment plays an important role in the outcome of anti-CD20 mAb therapy and that manipulation of this environment may improve the efficacy and produce long-term tumor control. The past few years have seen a surge of interest in immunomodulatory agents capable of overwriting immune suppressive networks into favorable clinical outcome. Currently, a number of such combinations with anti-CD20 mAb is under evaluation and some have produced encouraging outcomes in rituximab refractory disease. In this review, we give an outline of anti-CD20 mAbs and explore the combinations with immunomodulatory agents that enhance antitumor immunity through targeting stimulatory or inhibitory pathways and have proven potential to synergize with anti-CD20 mAb therapy. These agents, primarily mAbs, target CTLA-4, PD-1/PD-L1, and CD40.

Meeting report from the second EurocanPlatform summer school on translational cancer research, Portugal, 20-24 October 2014

The second EurocanPlatform summer school was held in Algarve, Portugal and attracted scientists, clinicians and pathologists with a common interest in cancer research to discuss the latest developments and challenges in the field. The meeting focused on translational cancer research and also included lectures, workshops and discussions, which covered all aspects of the translational research continuum, from early detection through treatment to survivorship. The rate of new cancer cases and cancer mortality increases every year. Although the last decade witnessed enormous progress in understanding cancer biology and the development of new therapies, the efficacy of these therapies is challenged by cancer resistance. It clearly suggests that new druggable targets are required and their translation from laboratory to bedside must be faster and more efficient to improve survival rates and standards of care.

Isolation of tumour antigen specific scFv's using a chimeric antigen receptor bicistronic retroviral vector in a mammalian screening protocol.

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.