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PURPOSE There is a need to refine the selection of patients with oropharyngeal squamous cell carcinoma (OPSCC) for treatment de-escalation. We investigated whether pretreatment absolute lymphocyte count (ALC) predicted overall survival (OS) benefit from the addition of concurrent chemotherapy to radical radiotherapy. PATIENTS AND METHODS This was an observational study of consecutive OPSCCs treated by curative-intent radiotherapy, with or without concurrent chemotherapy (n = 791) with external, independent validation from a separate institution (n = 609). The primary end point was OS at 5 years. Locoregional control (LRC) was assessed using competing risk regression as a secondary end point. Previously determined prognostic factors were used in a multivariable Cox proportional hazards model to assess the prognostic importance of ALC and the interaction between ALC and cisplatin chemotherapy use. RESULTS Pretreatment ALC was prognostic for 5-year OS on multivariable analysis (hazard ratio [HR] 0.64; 95% CI, 0.42 to 0.98; P = .04). It also predicted benefit from the use of concurrent cisplatin chemotherapy, with a significant interaction between cisplatin chemotherapy and pretreatment ALC (likelihood ratio test, P = .04): higher ALC count reduced the 5-year OS benefit compared with radiotherapy alone (HR 2.53; 95% CI, 1.03 to 6.19; P = .043). This was likely driven by an effect on LRC up to 5 years (interaction subdistribution HR 2.29; 95% CI, 0.68 to 7.71; P = .094). An independent validation cohort replicated the OS (HR 2.53; 95% CI, 0.98 to 6.52; P = .055) and LRC findings (interaction subdistribution HR 3.43; 95% CI, 1.23 to 9.52; P = .018). CONCLUSION For OPSCC, the pretreatment ALC is prognostic for OS and also predicts benefit from the addition of cisplatin chemotherapy to radiotherapy. These findings require prospective evaluation, and could inform the selection of good prognosis patients for a de-escalation trial.

Contains additional information methods data associated with accompanying supplemental figures, and additional data on in vivo studies, including information on experimental animals, housing and sample size. Contains 4 supplemental figures demonstrating: Figure 1, the cytotoxicity of clodronate liposomes against macrophages and tumor cells in vitro; Figure 2, that depletion of DC abrogates the priming of tumor-antigen specific CD8 T-cells following radiation and anti-CD40 mAb therapy; Figure 3, the impact of depletion of CD169 macrophages on radiation and anti-CD40 mAb therapy; Figure 4, combination radiation and R848 (TLR-7 agonist)leads to priming of tumor-specific CD8 T-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.

Concurrent but not sequential blockade of PD-1 is required to augment the efficacy of fractionated RT

Figure legends for Supplemental Figures 1-6

Treatment with FTY-720 has no direct effect on tumor growth and leads to deletion of non-LN resident and circulating T cells

Out-of-field tumor regression is a rare event following local single dose or fractionated RT.

Purpose: Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown.Experimental Design: The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field.Results: We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells.Conclusions: These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy. Clin Cancer Res; 23(18); 5514–26. ©2017 AACR.

The relative frequency of the top 25 clones from tumor 1 tracked in pre-therapy blood, post-therapy blood, and in tumor 2 post-therapy

Representative staining for PD-L1 on tumor cells (CD45-) and CD11b+Gr1+ cells

Gating strategy for tumor cells and tumor infiltrating immune cells

Supplemental figure 1: Gating strategy for determining tumor cell PD-L1 expression. Supplemental figure 2: Impact of low-dose fractionated RT on PD-L1 expression by CD11b+GR1Hi cells. Supplemental figure 3: Dual blockade of PD-1 and PD-L1 does not further enhance efficacy of RT combination. Supplemental figure 4: Blockade of PD-1 / PD-L1 in combination with local RT is well tolerated in mice. Supplemental figure 5: PD-1 / PD-L1 blockade does not directly sensitise tumor cells to RT. Supplemental figure 6: Scheduling of RT and anti-PD-L1 is critical for anti-tumor activity. Supplemental figure 7: Only acute administration of anti-PD-L1 mAb may be required to achieve efficacy when delivered in combination with RT.

Background: Ovarian cancer remains the most lethal gynaecological malignancy and there is an urgent need for the development of further therapies to improve patient survival. Tumour infiltrating lymphocyte (TIL) therapy has shown clear efficacy in immunogenic cancers, and TIL can be readily expanded ex vivo from samples of high grade serous ovarian cancer (HGSOC). Antibodies against checkpoint proteins are effective in increasing TIL yield and functional response from ovarian cancer TIL cultures, potentially increasing TIL product efficacy. However, it is unknown whether blockade of other key checkpoints, including programmed death ligand-1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) increase TIL yield in ex vivo cultures from HGSOC samples.Materials and Methods: TIL cultures were generated from fresh tissue samples and were incubated with CD3/CD28 Dynabeads® and IL-2. TIL were grown with or without the addition of 10µg/mL αPD-1, αTIM-3 or αLAG-3 antibodies as either single or repeated applications for 19 days. The phenotype of cultures was analysed by multiparameter flow cytometry. Interferon gamma (IFN-ᵧ) release in response to TIL co-culture with autologous tumour cultures or digests was measured by ELISA. Results: In keeping with previous findings the majority of TIL expressed at least one checkpoint protein. Specifically, TIL expressing PD-1 and TIM-3 retained a functional phenotype with co expression of OX40 and CD28. Expansion of TIL was increased following a single exposure to αPD-1 (1.20 fold) and αLAG-3 (1.31 fold) which was most marked in cultures with otherwise low proliferation. There was no additional increase in proliferation following repeated dosing. In co-culture with autologous tumour, TIL grown in the presence of checkpoint inhibitors showed increased IFN-ᵧ release suggesting increased functional activity in individual cultures. However, this response was not uniform across all cultures. Conclusions: These data suggests that initial blockade of checkpoint proteins is effective in increasing the yield of TIL expanded from HGSOC tumours, suggesting that checkpoint inhibition during TIL manufacture may be a useful method of increasing expansion efficiency.

Radiotherapy (RT) is a highly effective anti-cancer therapy delivered to around 50–60% of patients. It is part of therapy for around 40% of cancer patients who are cured of their disease. Until recently, the focus of this anti-tumour efficacy has been on the direct tumour cytotoxicity and RT-induced DNA damage. Recently, the immunomodulatory effects of RT on the tumour microenvironment have increasingly been recognized. There is now intense interest in potentially using RT to induce an anti-tumour immune response, which has led to rethinking into how the efficacy of RT could be further enhanced. Following the breakthrough of immune check point inhibitors (ICIs), a new era of immuno-oncology (IO) agents has emerged and established immunotherapy as a routine part of cancer treatment. Despite ICI improving outcomes in many cancer types, overall durable responses occur in only a minority of patients. The immunostimulatory effects of RT make combinations with ICI attractive to potentially amplify anti-tumour immunity resulting in increased tumour responses and improved outcomes. In contrast, tumours with profoundly immunosuppressive tumour microenvironments, dominated by myeloid-derived cell populations, remain a greater clinical challenge and RT may potentially further enhance the immunosuppression. To harness the full potential of RT and IO agent combinations, further insights are required to enhance our understanding of the role these immunosuppressive myeloid populations play, how RT influences these populations and how they may be therapeutically manipulated in combination with RT to improve outcomes further. These are exciting times with increasing numbers of IO targets being discovered and IO agents undergoing clinical evaluation. Multidisciplinary research collaborations will be required to establish the optimal parameters for delivering RT (target volume, dose and fractionation) in combination with IO agents, including scheduling to achieve maximal therapeutic efficacy.

Simple Summary Around 50% of all cancer patients receive radiotherapy as part of their treatment. Recently immunotherapy has shown promising results and become established as an effective treatment for some cancers. Combining radiotherapy and immunotherapy is a novel approach to further increase the number of patients responding to immunotherapy. Biological markers of response (biomarkers) are urgently required to hasten the clinical translation and improve outcomes further. Radiotherapy can both stimulate and inhibit the immune system and understanding the immune effects of radiotherapy on the tumour and surrounding cells may lead to the identification of predictive and prognostic biomarkers to help make more individualized treatment decisions, when combining radiotherapy with immunotherapy. This review summarizes the immune effects of radiotherapy and biomarkers of response identified to date; providing new perspectives for future research which may facilitate the development of novel radiotherapy immunotherapy combinations based on tumour immunology and biomarker identification. Abstract Radiotherapy (RT) is a highly effective anti-cancer treatment. Immunotherapy using immune checkpoint blockade (ICI) has emerged as a new and robust pillar in cancer therapy; however, the response rate to single agent ICI is low whilst toxicity remains. Radiotherapy has been shown to have local and systemic immunomodulatory effects. Therefore, combining RT and immunotherapy is a rational approach to enhance anti-tumour immune responses. However, the immunomodulatory effects of RT can be both immunostimulatory or immunosuppressive and may be different across different tumour types and patients. Therefore, there is an urgent medical need to establish biomarkers to guide clinical decision making in predicting responses or in patient selection for RT-based combination treatments. In this review, we summarize the immunological effects of RT on the tumour microenvironment and emerging biomarkers to help better understand the implications of these immunological changes, and we provide new insights into the potential for combination therapies with RT and immunotherapy.

Supplemental Digital Content is available in the text., Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells.

Purpose: Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown. Experimental Design: The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field. Results: We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells. Conclusions: These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy. Clin Cancer Res; 23(18); 5514–26. ©2017 AACR.

Cazaux et al. use intravital imaging to dissect anti-CD19 CAR T cell activity. This study uncovers both anatomical and functional diversity in the outcome of anti-CD19 CAR T cell interactions with tumor cells impacting engraftment, killing dynamics, and tumor immunoediting., CAR T cells represent a potentially curative strategy for B cell malignancies. However, the outcome and dynamics of CAR T cell interactions in distinct anatomical sites are poorly understood. Using intravital imaging, we tracked interactions established by anti-CD19 CAR T cells in B cell lymphoma–bearing mice. Circulating targets trapped CAR T cells in the lungs, reducing their access to lymphoid organs. In the bone marrow, tumor apoptosis was largely due to CAR T cells that engaged, killed, and detached from their targets within 25 min. Notably, not all CAR T cell contacts elicited calcium signaling or killing while interacting with tumors, uncovering extensive functional heterogeneity. Mathematical modeling revealed that direct killing was sufficient for tumor regression. Finally, antigen-loss variants emerged in the bone marrow, but not in lymph nodes, where CAR T cell cytotoxic activity was reduced. Our results identify a previously unappreciated level of diversity in the outcomes of CAR T cell interactions in vivo, with important clinical implications., Graphical 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.

Epithelial ovarian cancer (EOC) is the leading cause of gynaecological cancer-related death in Europe. Although most patients achieve an initial complete response with first-line treatment, recurrence occurs in more than 80% of cases. Thus, there is a clear unmet need for novel second-line treatments. EOC is frequently infiltrated with T lymphocytes, the presence of which has been shown to be associated with improved clinical outcomes. Adoptive T-cell therapy (ACT) using ex vivo-expanded tumour-infiltrating lymphocytes (TILs) has shown remarkable efficacy in other immunogenic tumours, and may represent a promising therapeutic strategy for EOC. In this preclinical study, we investigated the efficacy of using anti-CD3/anti-CD28 magnetic beads and IL-2 to expand TILs from freshly resected ovarian tumours. TILs were expanded for up to 3 weeks, and then subjected to a rapid-expansion protocol (REP) using irradiated feeder cells. Tumours were collected from 45 patients with EOC and TILs were successfully expanded from 89.7% of biopsies. Expanded CD4+ and CD8+ subsets demonstrated features associated with memory phenotypes, and had significantly higher expression of key activation and functional markers than unexpanded TILs. Expanded TILs produced anti-tumour cytokines when co-cultured with autologous tumour cells, inferring tumour cytotoxicity. Our findings demonstrate that it is possible to re-activate and expand tumour-reactive T cells from ovarian tumours. This presents a promising immunotherapy that could be used sequentially or in combination with current therapeutic strategies. Electronic supplementary material The online version of this article (10.1007/s00262-018-2211-3) contains supplementary material, which is available to authorized users.

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. Cancer Res; 74(19); 5458–68. ©2014 AACR.

The anti-CD20 mAb rituximab has revolutionized the treatment of B-cell malignancies, improving outcome for patients. Despite these improvements, the majority of patients still relapse and become refractory to rituximab. Further efforts to improve anti-CD20 mAb efficacy have recently focused on obinutuzumab /GA101, a novel anti-CD20 mAb glycoengineered to display enhanced Fc-mediated effector mechanisms and induce direct cell death.We provide an overview of the current insights into the mechanisms of action of obinutuzumab focusing on how structural modifications and differences to rituximab led to designation of obinutuzumab as a type II antibody. We summarize data from preclinical studies and recent clinical trials including the Phase III trial in chronic lymphocytic leukemia (CLL), which led to FDA approval in November 2013.Clinical data are now emerging confirming the promise of the initial preclinical data that demonstrated superior efficacy of obinutuzumab over rituximab at similar dosing. The emerging randomized Phase III data from older comorbid patients with previously untreated CLL demonstrated significant improvements in molecular remission rates and median progression-free survival of obinutuzumab plus chlorambucil versus rituximab plus chlorambucil. This emerging data provide reasons to be optimistic that outcomes for patients with B-cell malignancies can be further improved with obinutuzumab.

T cells engrafted with chimeric AgRs (CAR) are showing exciting potential for targeting B cell malignancies in early-phase clinical trials. To determine whether the second-generation CAR was essential for optimal antitumor activity, two CD28-based CAR constructs targeting CD19 were tested for their ability to redirect mouse T cell function against established B cell lymphoma in a BALB/c syngeneic model system. T cells armed with either CAR eliminated A20 B cell lymphoma in vivo; however, one construct induced a T cell dose-dependent acute toxicity associated with a raised serum Th1 type cytokine profile on transfer into preconditioned mice. Moreover, a chronic toxicity manifested as granuloma-like formation in spleen, liver, and lymph nodes was observed in animals receiving T cells bearing either CD28 CAR, albeit with different kinetics dependent upon the specific receptor used. This phenotype was associated with an expansion of CD4+CAR+ T cells and CD11b+Gr-1+ myeloid cells and increased serum Th2-type cytokines, including IL-10 and IL-13. Mouse T cells engrafted with a first-generation CAR failed to develop such autotoxicity, whereas toxicity was not apparent when T cells bearing the same receptors were transferred into C57BL/6 or C3H animals. In summary, the adoptive transfer of second-generation CD19-specific CAR T cells can result in a cell dose–dependent acute toxicity, whereas the prolonged secretion of high levels of Th2 cytokines from these CAR T cells in vivo drives a granulomatous reaction resulting in chronic toxicity. Strategies that prevent a prolonged Th2-cytokine biased CAR T cell response are clearly warranted.

Blockbuster antibody therapies have catapulted immune-based approaches to treat cancer into the consciousness of mainstay clinical research. On the back of this, other emerging immune-based therapies are providing great promise. T-cell therapy is one such area where recent trials using T cells genetically modified to express an antibody-based chimeric antigen receptor (CAR) targeted against the CD19 antigen have demonstrated impressive responses when adoptively transferred to patients with advanced chronic lymphocytic leukemia. The general concept of the CAR T cell was devised some 20 years ago. In this relatively short period of time, the technology to redirect T-cell function has moved at pace facilitating clinical translation; however, many questions remain with respect to developing the approach to improve CAR T-cell therapeutic activity and also to broaden the range of tumors that can be effectively targeted by this approach. This review highlights some of the underlying principles and compromises of CAR T-cell technology using the CD19-targeted CAR as a paradigm and discusses some of the issues that relate to targeting solid tumors with CAR T cells.

Adoptive cell therapy employing gene-modified T-cells expressing chimeric antigen receptors (CARs) has shown promising preclinical activity in a range of model systems and is now being tested in the clinical setting. The manufacture of CAR T-cells requires compliance with national and European regulations for the production of medicinal products. We established such a compliant process to produce T-cells armed with a first-generation CAR specific for carcinoembryonic antigen (CEA). CAR T-cells were successfully generated for 14 patients with advanced CEA(+) malignancy. Of note, in the majority of patients, the defined procedure generated predominantly CD4(+) CAR T-cells with the general T-cell population bearing an effector-memory phenotype and high in vitro effector function. Thus, improving the process to generate less-differentiated T-cells would be more desirable in the future for effective adoptive gene-modified T-cell therapy. However, these results confirm that CAR T-cells can be generated in a manner compliant with regulations governing medicinal products in the European Union.

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.

The second Cellular Therapy of Cancer Symposium, held in Milan, Italy last March, buoyed by the success of the Inaugural 2006 meeting in Manchester, UK, again brought together leading Wgures in cellular therapy research. The increase in delegate numbers registered in Milan surely reXected the rising interest in the Weld fuelled by recent high proWle publications demonstrating the success of adoptive cell therapy in cancer patients. The meeting was organized by members of the FP6 ATTACK consortium and hosted several scientiWc sessions spanning the research in cellular therapy in commercial and academic environment, the issues of TCR transfer and engineering and of redirected T-cell homing, subset representation and survival, and the latest pre and clinical development. The meeting also hosted a EU-sponsored talk given by Rosanna D’Amario, who summarized opportunities of career development within the FP7 People Programme. The meeting began with invited speakers from the commercial sector describing recent advances in the translation of laboratory studies to the clinical setting. Shahriar Yaghoubi (Stanford University School of Medicine, USA) described how adoptively transferred cells could be monitored in patients using PET imaging. Transferred cells can be imaged following direct labeling or through the use of imaging reporter genes such as the herpes simplex virus type 1 thymidine kinase PET reporter gene, which enable long-term visualization of therapeutic cell location, as well as survival or proliferation. Klaus Kuehlcke (Eufets AG, Germany) discussed GMP production of retroviruses, and methods for the expansion of therapeutic cells for clinical trials and Cliona Rooney (Baylor College of Medicine, USA) detailed how the Wilson Wolf bioreactors could be used to grow vast numbers of cells at densities of 8–11 £ 10 cells/cm. John Campbell (Miltentyi Biotech Ltd.) described how the company have generated “Peptivator” 15 amino acid peptide pools to generate antigen speciWc T-cells and Therese Croughs (Cytheris) presented data from several clinical trials using IL-7 to promote anti-tumor immune responses. The important role that cytokines and lymphodepleting chemotherapy can play in cellular therapy was discussed by several speakers. Charlie Surh (The Scripps Research Institute, USA) described how using cytokine/cytokine antibody complexes can reduce the eVective dose of cytokines required and can induce T-cell activation and non-lymphopenia induced homeostatic proliferation. Pamela Ohashi (Ontario Cancer Institute, Canada) presented data showing how IL-7 can enhance vaccine induced immune responses through multiple mechanisms such as the induction of Th17 cells, antagonization of TGF and inhibition of the negative regulator of T-cell activation, cbl-b. Luca Gattinoni (NCI, Bethesda, USA) and Reno Debets (Erasmus Medical Centre, The Netherlands) described how IL-21 could induce a more naive phenotype in ex vivo expanded T-cells and Luca demonstrated how this could be mimicked with a GSK3 inhibitor inducing T-cells with a naive/stem-cell like phenotype which were more potent in vivo. Michael Jensen (City of Hope National Medical Centre, USA) discussed how IL-15, in combination with selection for CD62L, leads to the generation of central memory T-cells which show enhanced engraftment in animal models. Host lymphodepletion has been shown to induce the proliferation and engraftment of transferred T-cells and Naomi Taylor (Institute de Genetique Moleculaire de Montpellier, J. S. Bridgeman · D. E. Gilham · R. E. Hawkins · E. J. Cheadle (&) Cellular Therapy Group, Department of Medical Oncology, The University of Manchester, Manchester, UK e-mail: echeadle@picr.man.ac.uk

Purpose: To study the production of chemokines by colorectal hepatic metastases.Experimental Design: Biopsies of resected colorectal hepatic metastases and nonneoplastic adjacent liver tissue were screened for chemokines using protein arrays and results were confirmed by ELISA and immunohistochemistry.Results: Two chemokines, eotaxin-2 and MCP-1, were found at elevated levels within the tumor biopsy compared with adjacent liver. The relative increase in expression from tumor was much higher for eotaxin-2 than MCP-1, with 10 of 25 donors having a >100-fold increase in expression compared with 0 of 24 donors for MCP-1. In a parallel analysis, eotaxin-2 was also found at elevated levels in the tumor region of primary colorectal cancer biopsies. Immunohistochemical staining indicated that carcinoembryonic antigen–positive tumor cells stained strongly for eotaxin-2, implicating these cells as the predominant source of the chemokine. In vitro studies confirmed that several colorectal tumor lines produce eotaxin-2 and that secretion of this chemokine could be depressed by IFN-γ and enhanced by the Th2-type cytokines interleukin-4 and interleukin-13. Jurkat T cells were engineered to express the receptor for eotaxin-2 (CCR3). These cells effectively migrated in response to eotaxin-2 protein, suggesting that immune cells gene modified to express a chemokine receptor may have improved abilities to home to tumor.Conclusions: Taken together, these observations confirm eotaxin-2 as a chemokine strongly associated with primary and metastatic tumors of colorectal origin. Furthermore, the importance of this result may be a useful tool in the development of targeted therapeutic approaches to colorectal tumors.

Strategies to augment anti-cancer immune responses have recently demonstrated therapeutic utility. To date clinical success has been achieved through targeting co-inhibitory checkpoints such as CTLA-4, PD-1, and PD-L1. However, approaches that target co-activatory pathways are also being actively being developed. Here we report that the novel TLR7-selective agonist DSR-29133 is well tolerated in mice and leads to acute immune activation. Administration of DSR-29133 leads to the induction of IFNα/γ, IP-10, TNFα, IL-1Ra and IL-12p70, and to a reduction in tumor burden in syngeneic models of renal cancer (Renca), metastatic osteosarcoma (LM8) and colorectal cancer (CT26). Moreover, we show that the efficacy of DSR-29133 was significantly improved when administered in combination with low-dose fractionated radiotherapy (RT). Effective combination therapy required weekly administration of DSR-29133 commencing on day 1 of a fractionated RT treatment cycle, whereas no enhancement of radiation response was observed when DSR-29133 was administered at the end of the fractionated RT cycle. Combined therapy resulted in curative responses in a high proportion of mice bearing established CT26 tumors which was dependent on the activity of CD8+ T-cells but independent of CD4+ T-cells and NK/NKT cells. Moreover, long-term surviving mice originally treated with DSR-29133 and RT were protected by a tumor-specific memory immune response which could prevent tumor growth upon rechallenge. These results demonstrate that DSR-29133 is a potent selective TLR7 agonist that when administered intravenously can induce anti-tumor immune responses that can be further enhanced through combination with low-dose fractionated RT.

After years of limited success, progress of anti-cancer immuno-therapeutics has been considerable over the past decade. Key to this progress has been the application of new biological insights around the importance and nature of immune checkpoints that are able to reverse down-regulation of anti-tumor immunity.An overview of the preclinical and recent clinical trial data on key immuno-regulatory agents currently in development, including antibody targeting of cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death receptor 1 (PD-1) on T-lymphocytes and its principal ligand (PD-L1) on tumor cells as well as immune agonists (e.g., anti-CD40).Durable long-term responses in some patients with advanced melanoma, initially with ipilimumab (anti-CTLA-4) and more recently antibodies targeting either PD-1 or PD-L1 in patients with melanoma and renal cancer, non-small-cell lung, bladder and head and neck cancers with less toxicity, have provided real optimism that immunotherapeutic approaches can improve outcomes in a wide range of cancer. The manageable tolerability of PD-1-pathway blockers and their unique mechanism of action are encouraging combination approaches. Current efforts focus on registration trials of single agents plus combinations in many different tumor types and treatment settings and identifying and developing predictive biomarkers of immunological response.

Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.

We have examined the potential of recombinant Escherichia coli expressing listeriolysin O (LLO) to deliver tumour antigens to dendritic cells (DCs) for cancer immunotherapy. Using OVA as a model tumour antigen, we have shown in murine DCs that E. coli expressing cytoplasmic LLO and OVA proteins can deliver the OVA K(b)-restricted epitope SIINFEKL for MHC class I presentation. In contrast, when E. coli expressing OVA alone were used, MHC class II presentation of the OVA 323-339 I-A(b)-restricted peptide was predominant. When injected in vivo, DCs pulsed with E. coli expressing LLO and OVA induced production of cytotoxic T-lymphocytes capable of lysing an OVA-expressing melanoma cell line (B16-OVA) and resulted in suppression of tumour growth following challenge with B16-OVA. Immunisation of mice by direct injection of E. coli LLO/OVA provided a more potent anti-tumour response, resulting in complete protection in 75% of mice. Injection of live bacteria was not necessary as immunisation with paraformaldehyde-fixed E. coli LLO/OVA provided an even stronger anti-tumour response against B16-OVA. Altogether, our data highlight the potential of this system as a novel and efficient strategy for tumour immunotherapy.

A link between microbial infection and cancer was first postulated by A. R. Ferguson in 1911 who described Schistosoma as the causative agent for bladder cancer in Egyptian men. Then, in 1926 the Nobel Prize for Medicine was awarded to Johannes Fibiger who identified a causal relationship between the nematode Spiroptera neoplastica and stomach cancer in rats. Although Fibiger's hypothesis was later to be disproved, a causative link between cancer and a range of infectious agents has subsequently been established.1 Nowadays there are a wide range of viruses and bacteria directly implicated in the transformation of normal cells and the eventual establishment of malignancy (Table 1). However, the link between infection and cancer is not always causative and the subject of this review is to describe a number of key advances in cancer immunotherapy based upon microbes. Many of these have already been translated into significant benefit for the patient and it is hoped that the latest advances in research will yield more effective and safer therapies and vaccines. Furthermore a number of therapies not initially designed to activate immune responses now appear to have a strong immune component involved in their mechanism of action. Table 1 A list of infectious agents known to be directly associated with the onset of specified malignancy

Over the last decade, the anti-CD20 monoclonal antibody (mAb) rituximab has substantially improved clinical outcome for patients with a range of B-cell malignancies, including non-Hodgkin lymphoma ...

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

The Chimeric Antigen Receptor (CAR) consists of an antibody-derived targeting domain fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR. CARs can potentially redirect the effector functions of a T-cell towards any protein and nonprotein target expressed on the cell surface as long as an antibody or similar targeting domain is available. This strategy thereby avoids the requirement of antigen processing and presentation by the target cell and is applicable to nonclassical T-cell targets like carbohydrates. This circumvention of HLA-restriction means that the CAR T-cell approach can be used as a generic tool broadening the potential of applicability of adoptive T-cell therapy. Proof-of-principle studies focusing upon the investigation of the potency of CAR T-cells have primarily focused upon the genetic modification of human and mouse T-cells for therapy. This chapter focuses upon methods to modify T-cells from both species to generate CAR T-cells for functional testing.

Monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell malignancies. Although Fc-dependent mechanisms of mAb-mediated tumor clearance have been extensively studied, the ability of mAbs to directly evoke programmed cell death (PCD) in the target cell and the underlying mechanisms involved remain under-investigated. We recently demonstrated that certain mAbs (type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD through an actin-dependent, lysosome-mediated process. Here, we reveal that the induction of PCD by these mAbs, including the type II anti-CD20 mAb GA101 (obinutuzumab), directly correlates with their ability to produce reactive oxygen species (ROS) in human B-lymphoma cell lines and primary B-cell chronic lymphocytic leukemia cells. ROS scavengers abrogated mAb-induced PCD indicating that ROS are required for the execution of cell death. ROS were generated downstream of mAb-induced actin cytoskeletal reorganization and lysosome membrane permeabilization. ROS production was independent of mitochondria and unaffected by BCL-2 overexpression. Instead, ROS generation was mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These findings provide further insights into a previously unrecognized role for NADPH oxidase-derived ROS in mediating nonapoptotic PCD evoked by mAbs in B-cell malignancies. This newly characterized cell death pathway may potentially be exploited to eliminate malignant cells, which are refractory to conventional chemotherapy and immunotherapy.

The Chimeric Antigen Receptor (CAR) consists of an antibody-derived targeting domain fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR. CARs can potentially redirect the effector functions of a T-cell towards any protein and nonprotein target expressed on the cell surface as long as an antibody or similar targeting domain is available. This strategy thereby avoids the requirement of antigen processing and presentation by the target cell and is applicable to nonclassical T-cell targets like carbohydrates. This circumvention of HLA-restriction means that the CAR T-cell approach can be used as a generic tool broadening the potential of applicability of adoptive T-cell therapy. Proof-of-principle studies focusing upon the investigation of the potency of CAR T-cells have primarily focused upon the genetic modification of human and mouse T-cells for therapy. This chapter focuses upon methods to modify T-cells from both species to generate CAR T-cells for functional testing.

T cells engineered to express chimeric receptors combining an external antibody binding domain with the CD3ζ T cell receptor (TCR) internal domain for triggering cell activation are being used for immunotherapeutic targeting of tumour cells in a non-HLA restricted manner. In this study we transduced T cells with a CD19-cTCR construct containing a truncated CD34 gene (tCD24) marker and used these to target the B cell antigen CD19 on the surface of a Hodgkin’s lymphoma (HL) cell line (L591) both in vitro and in vivo. Levels of tCD34 expression in transduced peripheral blood mononuclear cells ranged from 6-20% and this was increased to 82% after selection for trasnduced tCD34+ cells. In vitro cytotoxicity testing on a CD19+ HL cell line (L591) showed specific cell lysis initiated by the CD19-cTCR transduced PBMCs. In tumour prevention experiments severe combined immunodeficient (SCID) mice were inoculated with L591 HL cells subcutaneously (sc), and simultaneously inoculated with transduced CD19-cTCR T cells either sc or intravenously (iv). None of the 6 mice which received CD19-cTCR transduced T cells sc developed tumours compared to 4/6 (67%) iv inoculated and 17/22 (77%) un-inoculated controls (p=0.001). In tumour treatment experiments, transduced CD19-cTCR T cells were given iv to SCID mice 3-9 days after sc tumour formation. Whereas tumours progressed in all control uninoculated animals, complete tumour regression occurred with all treated mice surviving over 65 days (p=0.011). Time course experiments showed infiltrating CD19-cTCR transduced T cells (tCD34+) in tumour tissue at 11 and 18 days post inoculation, reaching a maximum of 41% of cells. These results suggest that CD19 could be a target for the immunotherapy of HL and that further preclinical studies are warranted.

T cells bearing chimeric antigen receptors (CARs) are broadly categorised into first- and second-generation receptors. Second-generation CARs contain a co-stimulatory signalling molecule and have been shown to secrete IL-2, undergo greater proliferation and to have enhanced persistence in vivo. However, we have shown that T cells bearing a first-generation CAR containing a CD19-targeting scFv (single-chain variable fragment) and the CD3ζ-signalling domain are able to produce IL-2 upon co-culture with CD19(+) B-cell lymphomas independent of CD28 activity. Here, we report that signalling through endogenous CD2 following ligation with its ligands, CD48 in mouse and CD58 in humans, drives IL-2 production by first-generation CD19-specific CAR. Moreover, the high levels of IL-2 produced by human T cells engrafted with a second-generation CD28-containing CAR during target-cell recognition are dependent to a degree upon CD2 receptor activity. These observations highlight the fact that the functional activity induced by T-cell-expressed CARs is dependent upon endogenous 'natural' receptor interactions. A deeper understanding of the role of these activities will serve to further refine the design of future CARs to either exploit or avoid these interactions.

The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

T cells gene-modified to express chimeric Ag receptors (CARs) have shown potent antitumor activity in vivo and are in clinical trials at locations worldwide. However, CAR activity has been investigated in mouse models in which Ag expression is restricted to the tumor. To explore the impact of normal tissue expression of the target Ag, we developed a mouse CD19-specific CAR to investigate antitumor efficacy against a syngeneic B cell lymphoma cell line within a background of normal CD19+ host B cells. Mouse T cells engrafted with the amCD19CD3ζ CAR specifically lysed A20 lymphoma targets and B cells in vitro. These T cells also eradicated a 12-d established disseminated A20 lymphoma in mice preconditioned with 6 Gy total body irradiation. In the short-term (7 d after adoptive transfer), amCD19z T cells underwent Ag-dependent proliferation in vivo with a concomitant depletion in host B cell levels. However, the levels of amCD19z CAR+ T cells decreased significantly at later time points, at which point host B cells returned, eventually reaching normal levels. In contrast, CAR+ T cells lacking a signaling domain or specificity for mCD19 persisted over extended periods in blood and spleen. Importantly, no overt clinical signs of autotoxicity were observed in tumor-free or tumor-bearing mice treated with amCD19z T cells over an extended period of time. These observations highlight the importance of studying the activity of CAR+ T cells in autologous models that have the normal range of tissue expression of Ag.

B-cell malignancies seem to be particularly amenable to immunotherapy and as such make particularly attractive targets for adoptive T-cell therapy. Murine T cells gene-modified to express a chimeric immune receptor specific for CD19+ (aCD19z) efficiently kill CD19 B-cell lymphoma cells in vitro. aCD19z T cells also secrete high levels of interleukin-2 during culture with target cells in a CD86 independent manner. aCD19z T cells proved effective at eradicating established B-cell lymphoma in a syngeneic model system when combined with a lymphodepleting preconditioning regimen. In mice deficient of T, B, and natural killer cells (severe combined immunodeficient/Beige), aCD19z T cells efficiently eradicated long-term (13 d) established tumors with 100% of treated animals remaining tumor free for greater than 77 days. Although gene-modified CD4+ and CD8+ were both active in this setting, poor engraftment by CD8+ T cells coupled with the rigorous expansion of CD4+ cells in the Balb/c background suggests that CD4+ T cells may be playing a predominant role in lymphoma rejection in this model. Taken together, the therapeutic effectiveness of aCD19z T cells in this model supports a recently opened phase 1 trial of this receptor in non-Hodgkin lymphoma.