RUBY® - a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties.

Despite the large number of existing bispecific antibody (bsAb) formats, the generation of novel bsAbs is still associated with development and bioprocessing challenges. Here, we present RUBY, a novel bispecific antibody format that allows rapid generation of bsAbs that fulfill key development criteria. The RUBYTM format has a 2 + 2 geometry, where two Fab fragments are linked via their light chains to the C-termini of an IgG, and carries mutations for optimal chain pairing. The unique design enables generation of bsAbs with mAb-like attributes. Our data demonstrate that RUBY bsAbs are compatible with small-scale production systems for screening purposes and can be produced at high yields (>3 g/L) from stable cell lines. The bsAbs produced are shown to, in general, contain low amounts of aggregates and display favorable solubility and stress endurance profiles. Further, compatibility with various IgG isotypes is shown and tailored Fc gamma receptor binding confirmed. Also, retained interaction with FcRn is demonstrated to translate into a pharmacokinetic profile in mice and non-human primates that is comparable to mAb controls. Functionality of conditional active RUBY bsAbs is confirmed in vitro. Anti-tumor effects in vivo have previously been demonstrated, and shown to be superior to a comparable mAb, and here it is further shown that RUBY bsAbs penetrate and localize to tumor tissue in vivo. In all, the RUBY format has attractive mAb-like attributes and offers the possibility to mitigate many of the development challenges linked to other bsAb formats, facilitating both high functionality and developability.

ATOR-1017 (evunzekibart), an Fc-gamma receptor conditional 4-1BB agonist designed for optimal safety and efficacy, activates exhausted T cells in combination with anti-PD-1.

BACKGROUND: 4-1BB (CD137) is a co-stimulatory receptor highly expressed on tumor reactive effector T cells and NK cells, which upon stimulation prolongs persistence of tumor reactive effector T and NK cells within the tumor and induces long-lived memory T cells. 4-1BB agonistic antibodies have been shown to induce strong anti-tumor effects that synergize with immune checkpoint inhibitors. The first generation of 4-1BB agonists was, however, hampered by dose-limiting toxicities resulting in suboptimal dose levels or poor agonistic activity. METHODS: ATOR-1017 (evunzekibart), a second-generation Fc-gamma receptor conditional 4-1BB agonist in IgG4 format, was designed to overcome the limitations of the first generation of 4-1BB agonists, providing strong agonistic effect while minimizing systemic immune activation and risk of hepatoxicity. The epitope of ATOR-1017 was determined by X-ray crystallography, and the functional activity was assessed in vitro and in vivo as monotherapy or in combination with anti-PD1. RESULTS: ATOR-1017 binds to a unique epitope on 4-1BB enabling ATOR-1017 to activate T cells, including cells with an exhausted phenotype, and NK cells, in a cross-linking dependent, FcγR-conditional, manner. This translated into a tumor-directed and potent anti-tumor therapeutic effect in vivo, which was further enhanced with anti-PD-1 treatment. CONCLUSIONS: These preclinical data demonstrate a strong safety profile of ATOR-1017, together with its potent therapeutic effect as monotherapy and in combination with anti-PD1, supporting further clinical development of ATOR-1017.

The Bispecific Tumor Antigen-Conditional 4-1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies.

4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.

Education, immigration and rising mental health inequality in Sweden.

Educational and income gradients in health are well established in the literature but there is need for a better understanding of how mental health inequalities change over time, and what drives the development. We aim to study how psychiatric diagnosis and its income-related inequality have changed over time in Sweden and to make a first attempt at disentangling the development by decomposing any changes in terms of changes in two important demographic characteristics: education and migration background. We use administrative patient data to study psychiatric inpatient diagnosis in the years 1994 and 2011. The study population comprises all individuals aged 31-64 years living in Sweden. Income-related inequalities are measured by the Concentration Index (CI). We decompose changes in the probability of receiving a diagnosis and changes in income-related inequality over time to understand the role of changing demographics. Our results show that over the study period the probability of receiving a psychiatric inpatient diagnosis increased by 12.6%, while the relative and absolute income-related inequalities in diagnosis increased by 48.2% and 66.7% respectively. In 2011, more than half of psychiatric inpatients were found among the poorest fifth of the population. The decomposition results suggest that changes in education and migration background have not played a substantial role in determining these increases. Education levels increased substantially over the study period which would be expected to protect against mental ill-health. Instead, we find that diagnoses have become more concentrated amongst the lowest educated individuals and the lowest income families, groups who appear to be increasingly disadvantaged. The growing proportion of individuals with foreign background in Sweden does, in fact, predict small increases in the probability of diagnosis, while the impact on diagnosis inequality varies depending on the definition of foreign background.

Inequalities in the economic consequences of depression and anxiety in Europe: a systematic scoping review.

BACKGROUND: Depression and anxiety are associated with adverse outcomes in educational achievements and economic performances. Moreover, the prevalence of these disorders is unequally distributed among different population subgroups. Our objective is to investigate whether the economic consequences of depression and anxiety differ between population subgroups of different gender, socioeconomic status (SES), ethnicity and age, in Europe. METHODS: A systematic scoping literature review was performed to identify studies where exposure to depression or anxiety was identified at baseline and consequences in education, sickness absence, disability pension, unemployment and income/earnings were measured at follow-up. RESULTS: Seventeen articles were included in this review and most of these were conducted in the Nordic countries. The consequences of depression and anxiety were stratified by gender in most of the articles. However, only in a few studies, the findings were stratified by SES, age and ethnicity. The negative consequences of depression in educational performance, disability pension and income are larger for men compared to women. Moreover, low SES individuals have more depression- and anxiety-related absence from work than high SES individuals. CONCLUSION: Our findings imply that the economic consequences of depression differ between population subgroups in Europe. This could have an impact on social stratification, shifting people who experience mental ill-health to lower SES groups or reinforcing an already disadvantaged position. More research is needed on unequal economic consequences of depression and anxiety in different population subgroups in Europe.

Child living arrangements following separation and mental health of parents in Sweden.

Father involvement and joint physical custody in post-separation families are increasingly common. In Sweden, 35 percent of the children of separated parents live equally much with both parents. Since parenthood is gendered, the associations between child living arrangement and parental health may vary between women and men. This study analyzes the association between children's living arrangement and mental health of parents, and how this interacts with material and social circumstances. Drawing on The Swedish Survey of Living Conditions (ULF) 2008-2013, the association between child living arrangements and mental health (worry/anxiety) of parents in five family structures: two biological parents, reconstituted with joint or main/sole custody arrangements, single with joint physical custody, and single with main or sole custody, were analyzed. Data on 9,225 mothers and fathers with resident children aged 0-17 were analyzed by logistic regressions for average marginal effects adjusting for socio-demographic, socio-economic and social factors. Analyses of interaction effects were made using the synergy index. Substantial family type differences were found in mental health between two biological parent family and all other family types for mothers, and two biological parent family and single parents for fathers. For the single mothers, the higher risk for worry and anxiety was still found following controls for socioeconomic factors. For fathers, the only differences that remained following control for socioeconomic factors was that of single fathers with children in joint physical custody. Interaction effects were found for the combination of single motherhood and non-employment, indicating a higher risk of mental health problems for single mothers (both with joint and sole custody), than would be expected from a simple addition of these exposures, suggesting that this is a vulnerable group. The results indicate that joint custody is associated with higher risk for worry and anxiety for the parents, especially for mothers both re-partnered and single, but also for single fathers.

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation.

BACKGROUND: The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. METHODS: ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. RESULTS: ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells. CONCLUSIONS: By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).

The effect of locally delivered cisplatin is dependent on an intact immune function in an experimental glioma model.

Several chemotherapeutic drugs are now considered to exert anti-tumour effects, by inducing an immune-promoting inflammatory response. Cisplatin is a potent chemotherapeutic agent used in standard medulloblastoma but not glioblastoma protocols. There is no clear explanation for the differences in clinical efficacy of cisplatin between medulloblastomas and glioblastomas, despite the fact that cisplatin is effective in vitro against the latter. Systemic toxicity is often dose limiting but could tentatively be reduced by intratumoral administration. We found that intratumoral cisplatin can cure GL261 glioma-bearing C57BL/6 mice and this effect was abolished in GL261-bearing NOD-scid IL2rγnull (NSG) mice. Contrary to previous results with intratumoral temozolomide cisplatin had no additive or synergistic effect with whole cell either GL261 wild-type or GM-CSF-transfected GL261 cells whole cell vaccine-based immunotherapy. While whole tumour cell immunizations increased CD8+ T-cells and decreased F4/80+ macrophages intratumorally, cisplatin had no effect on these cell populations. Taken together, our results demonstrate that intratumoral cisplatin treatment was effective with a narrow therapeutic window and may be an efficient approach for glioma or other brain tumour treatment.

Teleconsultation Using Mobile Phones for Diagnosis and Acute Care of Burn Injuries Among Emergency Physicians: Mixed-Methods Study.

BACKGROUND: The referral process in acute care remains challenging in many areas including burn care. Mobile phone apps designed explicitly for medical referrals and consultations could streamline the referral process by using structured templates and integrating features specific to different specialties. However, as these apps are competing with commercial chat services, usability becomes a crucial factor for successful uptake. OBJECTIVE: The aim of this study was to assess the usability of a mobile phone app for remote consultations and referrals of burn injuries. METHODS: A total of 24 emergency doctors and 4 burns consultants were recruited for the study. A mixed-methods approach was used including a usability questionnaire and a think-aloud interview. Think-aloud sessions were video-recorded, and content analysis was undertaken with predefined codes relating to the following 3 themes: ease of use, usefulness of content, and technology-induced errors. RESULTS: The users perceived the app to be easy to use and useful, but some problems were identified. Issues relating to usability were associated with navigation, such as scrolling and zooming. Users also had problems in understanding the meaning of some icons and terminologies. Sometimes, some users felt limited by predefined options, and they wanted to be able to freely express their clinical findings. CONCLUSIONS: We found that users faced problems mainly with navigation when the app did not work in the same way as the other apps that were frequently used. Our study also resonates with previous findings that when using standardized templates, the systems should also allow the user to express their clinical findings in their own words.

Family Structure, Child Living Arrangement and Mothers' Self-rated Health in Sweden-A Cross-Sectional Study.

Alternate living, i.e. children living 50-50 with their parents following separation is emerging as a new family form. This study is the first to differentiate separated mothers with sole/main custody from mothers with alternately living children, analysing health outcomes and using a sample representative of the population. The association between the self-rated health (SRH) of mothers and different family structures are examined. Parental cooperation is included in the analyses as a potential mediator. Data on 755 mothers from the 2010 Swedish Level of Living Survey were analyzed by multivariate logistic regression. Single mothers with sole/main custody reported poorer SRH than couple mothers in intact families while the difference was not significant for single mothers with children living alternately and mothers in stepfamilies. Controlling for potential confounders, probabilities for poor SRH for single mothers were reduced. The excess risk among mothers with sole/main custody may be due to poorer socioeconomic conditions. Employment was significantly more common among mothers with alternate living and an important explanatory factor for their better health compared to single mothers with sole/main custody. Adjusting for parental cooperation lowered the increased probability for poor SRH among single mothers with sole/main custody compared to single mothers with alternate living.

The discourses on induced abortion in Ugandan daily newspapers: a discourse analysis.

BACKGROUND: Ugandan law prohibits abortion under all circumstances except where there is a risk for the woman's life. However, it has been estimated that over 250 000 illegal abortions are being performed in the country yearly. Many of these abortions are carried out under unsafe conditions, being one of the most common reasons behind the nearly 5000 maternal deaths per year in Uganda. Little research has been conducted in relation to societal views on abortion within the Ugandan society. This study aims to analyze the discourse on abortion as expressed in the two main daily Ugandan newspapers. METHOD: The conceptual content of 59 articles on abortion between years 2006-2012, from the two main daily English-speaking newspapers in Uganda, was studied using principles from critical discourse analysis. RESULTS: A religious discourse and a human rights discourse, together with medical and legal sub discourses frame the subject of abortion in Uganda, with consequences for who is portrayed as a victim and who is to blame for abortions taking place. It shows the strong presence of the Catholic Church within the medial debate on abortion. The results also demonstrate the absence of medial statements related to abortion made by political stakeholders. CONCLUSIONS: The Catholic Church has a strong position within the Ugandan society and their stance on abortion tends to have great influence on the way other actors and their activities are presented within the media, as well as how stakeholders choose to convey their message, or choose not to publicly debate the issue in question at all. To decrease the number of maternal deaths, we highlight the need for a more inclusive and varied debate that problematizes the current situation, especially from a gender perspective.

Intratumorally implanted mesenchymal stromal cells potentiate peripheral immunotherapy against malignant rat gliomas.

Bone marrow-derived mesenchymal stromal cells (MSCs) target glioma extensions and micro-satellites efficiently when implanted intratumorally. Here, we report that intratumoral implantation of MSCs and peripheral immunotherapy with interferon-gamma (IFNγ) producing tumor cells improve the survival of glioma-bearing rats (54% cure rate) compared to MSC alone (0% cure rate) or immunotherapy alone (21% cure rate) by enforcing an intratumoral CD8(+) T cell response. Further analysis revealed that the MSCs up-regulate MHC classes I and II in response to IFNγ treatment in vitro and secrete low amounts of immunosuppressive molecules prostaglandin E2 and interleukin-10.

Immunizations with unmodified tumor cells and simultaneous COX-2 inhibition eradicate malignant rat brain tumors and induce a long-lasting CD8(+) T cell memory.

Malignant brain tumors induce pronounced immunosuppression, which diminishes immune responses generated by immunotherapy. Here we report that peripheral immunotherapy, using irradiated unmodified whole tumor cells, and systemic cyclooxygenase-2 inhibition induce cure in glioma-bearing rats (60% cure rate), whereas neither monotherapy was sufficient to cure any animal. Moreover, the combined therapy protected against secondary tumor challenges (89% cure rate) and the secondary immune response was correlated with increased plasma interferon-gamma levels and CD8(+) T cells systemically and intratumorally. In conclusion, we demonstrate that cyclooxygenase-2 inhibition is sufficient to render unmodified tumor cells immunogenic in immunotherapy of experimental brain tumors.

Family policy and inequalities in health in different welfare states.

This article focuses on differences in health and welfare outcomes for families with children in three European countries, discussed in relation to national policies for child and family welfare. Data consist of policy documents and cross-national surveys. The document analysis was based on policy documents that described government policies. The statistical analyses utilize data from the European Social Survey. For the analyses in this article, a sub-sample of child families was selected from the countries Slovenia, Sweden, and the United Kingdom. Data showed that England's policy has mainly addressed socially disadvantaged groups and areas. Sweden and Slovenia are mainly developing universal policies. The United Kingdom has high scores for subjective general health, but a steep income gradient in the population. Parents in England experience the highest level of at-risk-of-poverty. Sweden generally scores well on health outcomes and on level of at-risk-of-poverty, and the gradient in self-rated general health is the mildest. Slovenia has the weakest economy, but low levels of inequality and low child at-risk-for-poverty scores. The Slovenian example suggests that not only the level of economic wealth, but also its distribution in the population, has bearings on health and life satisfaction, not least on the health of children.

Intratumoral COX-2 inhibition enhances GM-CSF immunotherapy against established mouse GL261 brain tumors.

Immunotherapy has shown effectiveness against experimental malignant brain tumors, but the clinical results have been less convincing most likely due to immunosuppression. Prostaglandin E2 (PGE2 ) is the key immunosuppressive product of cyclooxygenase-2 (COX-2) and increased levels of PGE2 and COX-2 have been shown in several tumor types, including brain tumors. In the current study, we report enhanced cure rate of mice with established mouse GL261 brain tumors when immunized with granulocyte macrophage-colony stimulating factor (GM-CSF) secreting tumor cells and simultaneously treated with the selective COX-2 inhibitors parecoxib systemically (5 mg/kg/day; 69% cure rate) or valdecoxib intratumorally (5.3 µg/kg/day; 63% cure rate). Both combined therapies induced a systemic antitumor response of proliferating CD4(+) and CD8(+) T cells, and further analysis revealed T helper 1 (Th 1) cell supremacy. The GL261 tumor cell line produced low levels of PGE2 in vitro, and co-staining at the tumor site demonstrated that a large fraction of the COX-2(+) cells were derived from CD45(+) immune cells and more specifically macrophages (F4/80(+)), indicating that tumor-infiltrating immune cells constitute the primary source of COX-2 and PGE2 in this model. We conclude that intratumoral COX-2 inhibition potentiates GM-CSF immunotherapy against established brain tumors at substantially lower doses than systemic administration. These findings underscore the central role of targeting COX-2 during immunotherapy and implicate intratumoral COX-2 as the primary target.

Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion.

Despite temozolomide (TMZ) treatment, the prognosis for patients with glioblastoma multiforme is still dismal. As dose escalation of TMZ is limited by systemic toxicity, intratumoral delivery emerges as an attractive treatment modality, which may sustain cytotoxic drug concentrations intratumorally and induce immunogenic cell death. Both clinical and experimental gliomas have responded to immunotherapy, but the benefit of simultaneous chemo- and immunotherapy is inadequately studied. Here, we monitored survival of GL261-bearing C57BL/6 mice following a 3-day treatment with either intratumoral TMZ (micro-osmotic pump, 4.2 mg/kg/day) or systemic TMZ (i.p. injections, 50 mg/kg/day) alone, or combined with immunization using GM-CSF secreting GL261 cells. Peripheral and intratumoral leukocytes were analyzed by flow cytometry and immunohistochemistry. Intratumoral TMZ induced higher survival rate than systemic TMZ (45 vs. 8%). When T cells were depleted following intratumoral TMZ, the therapeutic effect was completely abrogated (0 % survival). Intratumoral TMZ synergistically increased survival rate of immunized mice (from 25 to 83%), while systemic TMZ failed (0%). While systemic TMZ induced a transient leukopenia, intratumoral TMZ and immunotherapy sustained the proliferation of CD8+ T cells and decreased the number of intratumoral immunosuppressive cells. In conclusion, intratumoral TMZ alone or in combination with immunotherapy could cure glioma-bearing mice, due to attenuation of local immunosuppression and increase in potential effector immune cells.

IFNγ in combination with IL-7 enhances immunotherapy in two rat glioma models.

Peripheral immunization, using a combination of interferon-gamma (IFNγ)- and interleukin-7 (IL-7)-producing tumor cells, eradicated 75% of pre-established intracerebral N32 rat glioma tumors, and prolonged survival in the more aggressive RG2 model. Rats immunized with IFNγ- and IL7-transduced N32 cells displayed increases in IFNγ plasma levels and proliferating circulating T cells when compared with rats immunized with N32-wild type cells. Following irradiation, the expression of MHC I and II was high on N32-IFNγ cells, but low on RG2-IFNγ cells. In conclusion, IFNγ and IL-7 immunizations prolong survival in two rat glioma models.

Inhibition of cyclooxygenase-2 enhances immunotherapy against experimental brain tumors.

Glioblastoma multiforme is the most common and aggressive malignant brain tumor in humans, and the prognosis is very poor despite conventional therapy. Immunotherapy represents a novel treatment approach, but the effect is often weakened by release of immune-suppressive molecules such as prostaglandins. In the current study, we investigated the effect of immunotherapy with irradiated interferon-γ (IFN-γ)-secreting tumor cells and administration of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib as treatment of established rat brain tumors. COX-2 inhibition and immunotherapy significantly enhanced the long-term cure rate (81% survival) compared with immunotherapy alone (19% survival), and there was a significant increase in plasma IFN-γ levels in animals treated with the combined therapy, suggesting a systemic T helper 1 immune response. COX-2 inhibition alone, however, did neither induce cure nor prolonged survival. The tumor cells were identified as the major source of COX-2 both in vivo and in vitro, and unmodified tumor cells produced prostaglandin E(2) in vitro, while the IFN-γ expressing tumor cells secreted significantly lower levels. In conclusion, we show that immunotherapy of experimental brain tumors is greatly potentiated when combined with COX-2 inhibition. Based on our results, the clinically available drug parecoxib may be added to immunotherapy against human brain tumors. Furthermore, the discovery that IFN-γ plasma levels can be used to determine the ongoing in vivo immune response has translational potential.