Striking an alliance between T cells and macrophages for enhanced cancer immunotherapy.

A new study by Yamada-Hunter et al. reveals a novel approach to promote synergy-rather than antagonism-between macrophages and engineered T cells, leading to enhanced antitumor immunity.

Optimization of manufacturing conditions for chimeric antigen receptor T cells to favor cells with a central memory phenotype.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells are genetically engineered to recognize tumor-associated antigens and have potent cytolytic activity against tumors. Adoptive therapy with CAR-T cells has been highly successful in B-cell leukemia and lymphoma. However, in solid tumor settings, CAR-T cells face a particularly hostile tumor microenvironment where multiple immune suppressive factors serve to thwart the anti-cancer immune response. Clinical trials of solid tumor antigen-targeted CAR-T cells have shown limited efficacy, and issues for current CAR-T cell therapies include failures of expansion and persistence, tumor entry, deletion and functional exhaustion. METHODS: We compared our standard protocol for CAR-T cell manufacturing, currently used to generate CAR-T cells for a phase 1 clinical trial, with two alternative approaches for T-cell activation and expansion. The resulting cultures were analyzed using multicolor flow cytometry, cytokine bead array and xCELLigence cytotoxicity assays. RESULTS: We have found that by changing the method of activation we can promote generation of CAR-T cells with enhanced CD62L and CCR7 expression, increased interleukin (IL)-2 production and retention of cytolytic activity, albeit with slower kinetics. DISCUSSION: We propose that these phenotypic characteristics are consistent with a central memory phenotype that will better enable CAR-T cell survival and persistence after activation in vivo, and we aim to test this in a continuation of our current phase 1 clinical trial of CAR-T cells in patients with advanced melanoma.

Phase I trial of Lipovaxin-MM, a novel dendritic cell-targeted liposomal vaccine for malignant melanoma.

INTRODUCTION: In this phase I study using a 3 + 3 dose escalation design, the safety, dose-limiting toxicity (DLT), immunogenicity and efficacy of intravenous Lipovaxin-MM-a multi-component dendritic cell-targeted liposomal vaccine against metastatic melanoma-was investigated. METHODS: Twelve subjects with metastatic cutaneous melanoma were recruited in three cohorts. Patients in Cohort A (n = 3) and Cohort B (n = 3) received three doses of 0.1 and 1 mL of Lipovaxin-MM, respectively, every 4 weeks. Patients in Cohort C (n = 6) received four doses of 3 mL vaccine weekly. Immunologic assessments of peripheral blood were made at regular intervals and included leukocyte subsets, cytokine levels, and Lipovaxin-MM-specific T-cell and antibody reactivities. Tumor responses were assessed by RECIST v1.0 at screening, then 8 weekly in Cohorts A and B and 6 weekly in Cohort C. RESULTS: Of a total of 94 adverse events (AEs) reported in ten subjects, 43 AEs in six subjects were considered to be possibly or probably vaccine-related. Most (95%) vaccine-related AEs were grade 1 or 2, two (5%) grade 3 vaccine-related AEs of anemia and lethargy were recorded, and higher grade AEs and DLTs were not observed. No consistent evidence of vaccine-specific humoral or cellular immune responses was found in post-immunization blood samples. One patient had a partial response, two patients had stable disease, and the remaining patients had progressive disease. CONCLUSIONS: Lipovaxin-MM was well tolerated and without clinically significant toxicity. Immunogenicity of Lipovaxin-MM was not detected. Partial response and stable disease were observed in one and two patients, respectively.

Logic-gated approaches to extend the utility of chimeric antigen receptor T-cell technology.

Chimeric antigen receptor (CAR)-T cell therapy has been clinically validated as a curative treatment for the difficult to treat malignancies of relapsed/refractory B-cell acute lymphoblastic leukaemia and lymphoma. Here, the CAR-T cells are re-directed towards a single antigen, CD19, which is recognised as a virtually ideal CAR target antigen because it has strong, uniform expression on cancer cells, and is otherwise expressed only on healthy B cells, which are 'dispensable'. Notwithstanding the clinical success of CD19-CAR-T cell therapy, its single specificity has driven therapeutic resistance in 30% or more of cases with CD19-negative leukaemic relapses. Immune checkpoint blockade is also a highly successful cancer immunotherapeutic approach, but it will be less useful for many patients whose malignancies either lack a substantial somatic mutation load or whose tumours are intrinsically resistant. Although CAR-T cell therapy could serve this unmet medical need, it is beset by several major limitations. There is a lack of candidate antigens that would satisfy the requirements for ideal CAR targets. Biological properties such as clonal heterogeneity and micro-environmental conditions hostile to T cells are inherent to many solid tumours. Past clinical studies indicate that on-target, off-tumour toxicities of CAR-T cell therapy may severely hamper its application. Therefore, re-designing CARs to increase the number of antigen specificities recognised by CAR-T cells will broaden tumour antigen coverage, potentially overcoming tumour heterogeneity and limiting tumour antigen escape. Tuning the balance of signalling within bi-specific CAR-T cells may enable tumour targeting while sparing normal tissues, and thus minimise on-target, off-tumour toxicities.

GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

DNA vaccines encoding membrane-bound or secreted forms of heat shock protein 70 exhibit improved potency.

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage-associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane-bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag-specific T-cell responses. Our results demonstrated the ability of membrane-bound and secreted HSP70 to significantly enhance gag-specific T-cell responses and increase the breadth of T-cell responses to include subdominant epitopes. Membrane-bound or secreted HSP70 also significantly improved the multifunctionality of HIV-specific T cells and T-cell proliferation, which is important for maintaining T-cell integrity. Most importantly, the inclusion of membrane-bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.

Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells are a novel immunotherapy for cancer. To achieve anti-tumor efficacy, these cells must survive, expand, and persist after infusion into patients, functions that are reportedly best achieved by cells with a stem or central-memory rather than effector-memory phenotype. We have developed third-generation CAR T cells specific for the tumor-associated antigen GD2 for use in a phase I clinical trial. We investigated the optimal cell culture conditions for CAR T-cell production, and here we describe the relative effects of 3 activation and cytokine conditions on CAR T-cell expansion, effector function and phenotype. METHODS: Peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 or anti-CD3 and Retronectin. Activated cells were transduced with the CAR-encoding retroviral vector and expanded in either interleukin (IL)-2 or IL-7 and IL-15. Immune phenotype and expansion were tracked throughout the culture, and transduction efficiency, and subsequent GD2-specific effector functions were evaluated by flow cytometry and cytotoxic T lymphocytes assay. RESULTS: CD3/Retronectin stimulation with IL-2 resulted in poorer activation, expansion and Th1 cytokine secretion of CAR T cells than CD3/CD28 stimulation with either IL-2 or IL-7 and IL-15. However, CAR T cells cultured in CD3/CD28/IL7/IL-15 and CD3/Retronectin/IL-2 had superior cytotoxic T lymphocyte activity and a more stem-like phenotype. DISCUSSION: The combination of CD3 and CD28 with IL-7 and IL-15 gave the best balance of CAR T-cell expansion and potent GD2-specific effector functions while retaining a stem/memory phenotype, and these growth conditions will therefore be used to manufacture CAR T cells for our phase I clinical trial.

Induction of antigen-positive cell death by the expression of perforin, but not DTa, from a DNA vaccine enhances the immune response.

The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

Safety and biological outcomes following a phase 1 trial of GD2-specific CAR-T cells in patients with GD2-positive metastatic melanoma and other solid cancers.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapies specific for the CD19 and B-cell maturation antigen have become an approved standard of care worldwide for relapsed and refractory B-cell malignancies. If CAR-T cell therapy for non-hematological malignancies is to achieve the same stage of clinical development, then iterative early-phase clinical testing can add value to the clinical development process for evaluating CAR-T cell products containing different CAR designs and manufactured under differing conditions. METHODS: We conducted a phase 1 trial of third-generation GD2-specific CAR-T cell therapy, which has previously been tested in neuroblastoma patients. In this study, the GD2-CAR-T therapy was evaluated for the first time in metastatic melanoma patients in combination with BRAF/MEK inhibitor therapy, and as a monotherapy in patients with colorectal cancer and a patient with fibromyxoid sarcoma. Feasibility and safety were determined and persistence studies, multiplex cytokine arrays on sera and detailed immune phenotyping of the original CAR-T products, the circulating CAR-T cells, and, in select patients, the tumor-infiltrating CAR-T cells were performed. RESULTS: We demonstrate the feasibility of manufacturing CAR-T products at point of care for patients with solid cancer and show that a single intravenous infusion was well tolerated with no dose-limiting toxicities or severe adverse events. In addition, we note significant improvements in CAR-T cell immune phenotype, and expansion when a modified manufacturing procedure was adopted for the latter 6 patients recruited to this 12-patient trial. We also show evidence of CAR-T cell-mediated immune activity and in some patients expanded subsets of circulating myeloid cells after CAR-T cell therapy. CONCLUSIONS: This is the first report of third-generation GD2-targeting CAR-T cells in patients with metastatic melanoma and other solid cancers such as colorectal cancer, showing feasibility, safety and immune activity, but limited clinical effect. TRIAL REGISTRATION NUMBER: ACTRN12613000198729.

Endogenous bystander killing mechanisms enhance the activity of novel FAP-specific CAR-T cells against glioblastoma.

Objectives: CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. Methods: We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function in vitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). Results: FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. Conclusions: Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

A Poisson distribution-based general model of cancer rates and a cancer risk-dependent theory of aging.

This article presents a formula for modeling the lifetime incidence of cancer in humans. The formula utilizes a Poisson distribution-based "np" model to predict cancer incidence, with "n" representing the effective number of cell turnover and "p" representing the probability of single-cell transformation. The model accurately predicts the observed incidence of cancer in humans when a reduction in cell turnover due to aging is taken into account. The model also suggests that cancer development is ultimately inevitable. The article proposes a theory of aging based on this concept, called the "np" theory. According to this theory, an organism maintains its order by balancing cellular entropy through continuous proliferation. However, cellular "information entropy" in the form of accumulated DNA mutations increases irreversibly over time, restricting the total number of cells an organism can generate throughout its lifetime. When cell division slows down and fails to compensate for the increased entropy in the system, aging occurs. Essentially, aging is the phenomenon of running out of predetermined cell resources. Different species have evolved separate strategies to utilize their limited cell resources throughout their life cycle.

Targeting the dendritic cell-T cell axis to develop effective immunotherapies for glioblastoma.

Glioblastoma is an aggressive primary brain tumor that has seen few advances in treatments for over 20 years. In response to this desperate clinical need, multiple immunotherapy strategies are under development, including CAR-T cells, immune checkpoint inhibitors, oncolytic viruses and dendritic cell vaccines, although these approaches are yet to yield significant clinical benefit. Potential reasons for the lack of success so far include the immunosuppressive tumor microenvironment, the blood-brain barrier, and systemic changes to the immune system driven by both the tumor and its treatment. Furthermore, while T cells are essential effector cells for tumor control, dendritic cells play an equally important role in T cell activation, and emerging evidence suggests the dendritic cell compartment may be deeply compromised in glioblastoma patients. In this review, we describe the immunotherapy approaches currently under development for glioblastoma and the challenges faced, with a particular emphasis on the critical role of the dendritic cell-T cell axis. We suggest a number of strategies that could be used to boost dendritic cell number and function and propose that the use of these in combination with T cell-targeting strategies could lead to successful tumor control.

Genome-wide identification of human FOXP3 target genes in natural regulatory T cells.

The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3(+) Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25(+)FOXP3(+) Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.

Characterising Distinct Migratory Profiles of Infiltrating T-Cell Subsets in Human Glioblastoma.

Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cell-based immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients. However, a major challenge for T-cell-based immunotherapy of glioblastoma and other solid cancers is T-cell infiltration into tumours. This process is mediated by chemokine-chemokine receptor and integrin-adhesion molecule interactions, yet the specific nature of the molecules that may facilitate T-cell homing into glioblastoma are unknown. Here, we have characterised chemokine receptor and integrin expression profiles of endogenous glioblastoma-infiltrating T cells, and the chemokine expression profile of glioblastoma-associated cells, by single-cell RNA-sequencing. Subsequently, chemokine receptors and integrins were validated at the protein level to reveal enrichment of receptors CCR2, CCR5, CXCR3, CXCR4, CXCR6, CD49a, and CD49d in glioblastoma-infiltrating T-cell populations relative to T cells in matched patient peripheral blood. Complementary chemokine ligand expression was then validated in glioblastoma biopsies and glioblastoma-derived primary cell cultures. Together, enriched expression of homing receptor-ligand pairs identified in this study implicate a potential role in mediating T-cell infiltration into glioblastoma. Importantly, our data characterising the migratory receptors on endogenous tumour-infiltrating T cells could be exploited to enhance the tumour-homing properties of future T-cell immunotherapies for glioblastoma.

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma.

BACKGROUND: Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited. METHODS: We profiled an extensive biobank of patients' biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. RESULTS: Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. CONCLUSIONS: Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

Potent Stimulation of the Androgen Receptor Instigates a Viral Mimicry Response in Prostate Cancer.

Inhibiting the androgen receptor (AR), a ligand-activated transcription factor, with androgen deprivation therapy is a standard-of-care treatment for metastatic prostate cancer. Paradoxically, activation of AR can also inhibit the growth of prostate cancer in some patients and experimental systems, but the mechanisms underlying this phenomenon are poorly understood. This study exploited a potent synthetic androgen, methyltestosterone (MeT), to investigate AR agonist-induced growth inhibition. MeT strongly inhibited growth of prostate cancer cells expressing AR, but not AR-negative models. Genes and pathways regulated by MeT were highly analogous to those regulated by DHT, although MeT induced a quantitatively greater androgenic response in prostate cancer cells. MeT potently downregulated DNA methyltransferases, leading to global DNA hypomethylation. These epigenomic changes were associated with dysregulation of transposable element expression, including upregulation of endogenous retrovirus (ERV) transcripts after sustained MeT treatment. Increased ERV expression led to accumulation of double-stranded RNA and a "viral mimicry" response characterized by activation of IFN signaling, upregulation of MHC class I molecules, and enhanced recognition of murine prostate cancer cells by CD8+ T cells. Positive associations between AR activity and ERVs/antiviral pathways were evident in patient transcriptomic data, supporting the clinical relevance of our findings. Collectively, our study reveals that the potent androgen MeT can increase the immunogenicity of prostate cancer cells via a viral mimicry response, a finding that has potential implications for the development of strategies to sensitize this cancer type to immunotherapies. Significance: Our study demonstrates that potent androgen stimulation of prostate cancer cells can elicit a viral mimicry response, resulting in enhanced IFN signaling. This finding may have implications for the development of strategies to sensitize prostate cancer to immunotherapies.

Erratum: Clinical chimeric antigen receptor T-cell therapy: a new and promising treatment modality for glioblastoma.

[This corrects the article DOI: 10.1002/cti2.1050.].

Effects of Chemotherapy Agents on Circulating Leukocyte Populations: Potential Implications for the Success of CAR-T Cell Therapies.

Adoptive T-cell therapy using autologous T cells genetically modified to express cancer-specific chimeric antigen receptors (CAR) has emerged as a novel approach for cancer treatment. CAR-T cell therapy has been approved in several major jurisdictions for treating refractory or relapsed cases of B-cell precursor acute lymphoblastic leukaemia and diffuse large B-cell lymphoma. However, in solid cancer patients, several clinical studies of CAR-T cell therapy have demonstrated minimal therapeutic effects, thus encouraging interest in better integrating CAR-T cells with other treatments such as conventional cytotoxic chemotherapy. Increasing evidence shows that not only do chemotherapy drugs have tumoricidal effects, but also significantly modulate the immune system. Here, we discuss immunomodulatory effects of chemotherapy drugs on circulating leukocyte populations, including their ability to enhance cytotoxic effects and preserve the frequency of CD8+ T cells and to deplete immunosuppressive populations including regulatory T cells and myeloid-derived suppressor cells. By modulating the abundance and phenotype of leukocytes in the blood (the 'raw material' for CAR-T cell manufacturing), we propose that prior chemotherapy could facilitate production of the most effective CAR-T cell products. Further research is required to directly test this concept and identify strategies for the optimal integration of CAR-T cell therapies with cytotoxic chemotherapy for solid cancers.

The Role of Cytokines and Chemokines in Shaping the Immune Microenvironment of Glioblastoma: Implications for Immunotherapy.

Glioblastoma is the most common form of primary brain tumour in adults. For more than a decade, conventional treatment has produced a relatively modest improvement in the overall survival of glioblastoma patients. The immunosuppressive mechanisms employed by neoplastic and non-neoplastic cells within the tumour can limit treatment efficacy, and this can include the secretion of immunosuppressive cytokines and chemokines. These factors can play a significant role in immune modulation, thus disabling anti-tumour responses and contributing to tumour progression. Here, we review the complex interplay between populations of immune and tumour cells together with defined contributions by key cytokines and chemokines to these intercellular interactions. Understanding how these tumour-derived factors facilitate the crosstalk between cells may identify molecular candidates for potential immunotherapeutic targeting, which may enable better tumour control and improved patient survival.

Positron Emission Tomographic Imaging of Tumor Cell Death Using Zirconium-89-Labeled APOMAB® Following Cisplatin Chemotherapy in Lung and Ovarian Cancer Xenograft Models.

PURPOSE: Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. PROCEDURES: In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [89Zr]ZrIV. Tumor responses were monitored using bioluminescence imaging and caliper measurements. [89Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. RESULTS: Tumor uptake of [89Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. CONCLUSION: Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.