key: cord-0989534-la7azktf
authors: Karakike, E.; Dalekos, G. N.; Koutsodimitropoulos, I.; Saridaki, M.; Pourzitaki, C.; Papathanakos, G.; Kotsaki, A.; Chalvatzis, S.; Dimakopoulou, V.; Vechlidis, N.; Paramythiotou, E.; Avgoustou, C.; Ioakeimidou, A.; Kouriannidi, E.; Komnos, A.; Neou, E.; Rovina, N.; Stefanatou, E.; Milionis, H.; Nikolaidis, G.; Koutsoukou, A.; Damoraki, G.; Dimopoulos, G.; Zoumpos, V.; Eugen-Olsen, J.; Akinosoglou, K.; Gatselis, N. K.; Koulouras, V.; Gkeka, E.; Markou, N.; Netea, M. G.; Giamarellos-Bourboulis, E. J.
title: ESCAPE: An Open-Label Trial of Personalized Immunotherapy in Critically Ill COVID-19 Patients
date: 2021-01-26
journal: nan
DOI: 10.1101/2021.01.20.21250182
sha: 3f9271b9a6c51e30ee32b327609b4e7f78c895cd
doc_id: 989534
cord_uid: la7azktf

ABSTRACT Rationale Macrophage activation syndrome (MAS) and complex immune dysregulation (CID) often underlie acute respiratory distress (ARDS) in COVID-19. Objective To investigate the outcome of personalized immunotherapy in critical COVID-19. Methods In this open-label prospective trial, 102 patients with SOFA (sequential organ failure assessment) score [≥]2 or ARDS by SARS-CoV-2 were screened for MAS (ferritin more than 4420 ng/ml) and CID (ferritin [≤]4420 ng/ml and low expression of HLA-DR on CD14-monocytes). Patients with MAS and CID with increased aminotransferases were assigned to intravenous anakinra; those with CID and normal aminotransferases to tocilizumab. The primary outcome was at least 25% decrease of SOFA score and/or 50% increase of respiratory ratio by day 8; 28-day mortality, change of SOFA score by day 28; serum biomarkers and cytokine production by mononuclear cells were secondary endpoints. Measurements and Main Results The primary study endpoint was met in 58.3% of anakinra-treated patients and in 33.3% of tocilizumab-treated patients (odds ratio 3.11; 95% CIs 1.29-7.73; P: 0.011). No differences were found in mortality and in SOFA score changes. By day 4, ferritin was decreased among anakinra-treated patients; interleukin (IL)-6, soluble urokinase plasminogen activator receptor (suPAR) and the expression of HLA-DR were increased among tocilizumab-treated patients. Anakinra increased capacity of mononuclear cells to produce IL-6. Survivors by day 28 who received anakinra were distributed to scales of the WHO clinical progression of lower severity. Greater incidence of secondary infections was found with tocilizumab treatment. Conclusions Biomarkers may guide favourable anakinra responses in critically ill patients with COVID-19. Trial Registration: ClinicalTrials.gov, NCT04339712 Key-words: anakinra; tocilizumab; acute respiratory distress syndrome; COVID-19; interleukin-6; ferritin; HLA-DR; macrophage activation; monocytes Abstract Word count: 250

The study was funded in part by the Hellenic Institute for the Study of Sepsis and in part by the Horizon 2020 grant RISKinCOVID.

EJGB conceptualized the study design, participated in data analysis and drafting the manuscript, had full access to all of the study data and takes responsibility for their 

ESCAPE was an open-label phase II trial where critically ill patients with were allocated to treatment with anakinra or tocilizumab based on immune function tests. Findings suggest most clinical benefit among critically ill patients treated with anakinra when ferritin is more than 4420 ng/ml or when a combination of low expression of HLA-DR on CD14-circulating monocytes and increase of liver aminotransferases is found.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Objective To investigate the outcome of personalized immunotherapy in critical Methods In this open-label prospective trial, 102 patients with SOFA (sequential organ failure assessment) score ≥2 or ARDS by SARS-CoV-2 were screened for MAS (ferritin more than 4420 ng/ml) and CID (ferritin ≤4420 ng/ml and low expression of HLA-DR on CD14-monocytes). Patients with MAS and CID with increased aminotransferases were assigned to intravenous anakinra; those with CID and normal aminotransferases to tocilizumab. The primary outcome was at least 25% decrease of SOFA score and/or 50% increase of respiratory ratio by day 8; 28-day mortality, change of SOFA score by day 28; serum biomarkers and cytokine production by mononuclear cells were secondary endpoints.

The primary study endpoint was met in 58.3% of anakinra-treated patients and in 33.3% of tocilizumab-treated patients (odds ratio 3.11; 95% CIs 1.29-7.73; P: 0.011). No differences were found in mortality and in SOFA score changes. By day 4, ferritin was decreased among anakinra-treated patients; interleukin (IL)-6, soluble urokinase plasminogen activator receptor (suPAR) and the expression of HLA-DR were increased among tocilizumab-treated patients.

Anakinra increased capacity of mononuclear cells to produce IL-6. Survivors by day 28 who received anakinra were distributed to scales of the WHO clinical progression of lower severity. Greater incidence of secondary infections was found with tocilizumab treatment.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint

Early from the start of the pandemic caused by SARS-CoV-2, it was realized that patients with severe or critical illness present with profound immune dysregulation with the main features of lymphopenia, hyper-production of proinflammatory cytokines, hyper-ferritinemia and pro-inflammatory/anti-inflammatory imbalance (1) (2) (3) (4) . An important question arising regarding the transition from severe COVID-19 pneumonia to critical illness and acute respiratory distress syndrome (ARDS) was whether this was identical in all patients, or whether different immune endotypes can be described in the severely ill patients (5) . Based on earlier studies, we used serum ferritin and the expression of the human leukocyte antigen (HLA)-DR on circulating monocytes as biomarkers of immune dysregulation. We selected a cutoff ferritin of 4,420 ng/ml for the diagnosis of macrophage activation syndrome (MAS) as previously done for sepsis (6, 7) . Analysis showed that almost 25% of patients with ARDS by COVID-19 had MAS, whereas the rest had low expression of HLA-DR on circulating monocytes. This low HLA-DR expression was interpreted as complex immune dysregulation (CID), since circulating monocytes retained their potential for cytokine production (5) .

Previous studies in sepsis have shown that patients with MAS have survival benefit from treatment with anakinra, a non-glycosylated recombinant protein of interleukin (IL)-1 receptor antagonist (8) . The addition of the IL-6 receptor antagonist tocilizumab in cultures of circulating monocytes of patients with ARDS by and CID restored the expression of HLA-DR (5). These findings guided the concept to approach immunotherapy in critically ill patients with COVID-19 through personalized treatment based on their immune profile, i.e. MAS or CID. ESCAPE (Efficiency in management of organ dysfunction associated with infection by the . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. Enrolled patients were adults of both genders with written informed consent provided by legal representatives; organ dysfunction; and laboratory findings of MAS or CID. Organ dysfunction was defined as either total SOFA (sequential organ failure assessment) score ≥2 or ARDS. MAS was defined as any serum ferritin greater than 4,420ng/ml. CID was defined as serum ferritin ≤4,420ng/ml and less than 5,000 receptors of the membrane HLA-DR or 30 MFI (mean fluorescence intensity units) of HLA-DR on blood CD14-monocytes by flow cytometry (see online supplement for Methods details). Exclusion criteria were: stage IV malignancy; do not resuscitate decision; active tuberculosis; infection by the human immunodeficiency virus; primary immunodeficiencies; intake of corticosteroids for more 15 days; anti-cytokine biological treatments the last one month; history of systemic lupus erythematosus or demyelinating disorder; and pregnancy or lactation.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint 8 Patients were treated with anakinra for MAS and with tocilizumab for CID.

Patients with CID and neutrophil count < 2,500/mm 3 or platelet count < 100,000/mm 3 or serum aminotransferases more than 1.5 x the upper normal when tocilizumab is contraindicated were treated with anakinra. Both drugs were administered intravenously; tocilizumab as single 8mg/kg dose up to 800mg maximum; and anakinra 200 mg every eight hours for seven days; anakinra was adjusted to 100 mg every eight hours for seven days for patients with creatinine clearance less than 30 ml/min. Peripheral blood mononuclear cells (PBMCs) were isolated at baseline and on day 4 and stimulated for cytokine production. Patients were followed-up daily; adverse events were captured (online supplement).

The primary study endpoint was at least 25% decrease of baseline SOFA score and/or at least 50% increase of the baseline PaO 2 /FiO 2 ratio by day 8.

Secondary study endpoints were 28-day mortality; change of SOFA score by day 28;

and changes of serum biomarkers and of cytokine production by PBMCs. Exploratory endpoints were the WHO clinical progression scale (CPS) (9) and the length of hospital stay.

Concurrent comparators with ARDS hospitalized at three other ICUs of Greek tertiary hospitals were analysed (online supplement). The primary endpoint was compared between anakinra-treated and tocilizumab-treated patients by the Fisher exact test; odds ratio (OR) and 95% confidence intervals (CIs) were calculated according to Mantel and Haenszel. The primary endpoint was validated by forward stepwise logistic regression analysis. Biomarkers and cytokines were expressed as means ± SE; serial comparisons were done by the Wilcoxon's paired test and correlations according to Spearman's rank of order. The distribution of the WHO CPS . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint was compared by the Pearson's chi-square test. The time to hospital discharge was compared by the log-rank test. Any value of P below 0.05 was considered significant.

The study was exploratory and no power calculation was done.

The first patient was enrolled on April 2 nd 2020 and the last patient on November 16 th 2020. A total of 102 patients were enrolled; 42 were allocated to treatment with tocilizumab and 60 to treatment with anakinra. Among anakinratreated patients, 14 had MAS and 46 had CID with increased aminotransferases ( Figure 1 ). No differences were found between patients allocated to anakinra treatment and to tocilizumab treatment regarding their baseline characteristics (Table   1) .

A total of 84 concurrent comparators were used. These were hospitalized during the same time period in three other ICUs of tertiary hospitals. They were selected from a database of 136 patients after applying the inclusion and exclusion criteria of the ESCAPE trial with the exception of the laboratory criteria for CID which were not available (supplementary Figure 1 ).

The primary study endpoint was met in 58.3% of anakinra-treated patients and in 33.3% of tocilizumab-treated patients ( Table 2) . This difference was confirmed after stepwise forward logistic regression analysis (OR 3.11; 95% CIs 1.29-7.73; P: Table 3 ). However, no difference was found between the two groups regarding 28-day mortality and the change of SOFA score by day 28 (Table 2 ).

Immunotherapy was accompanied by changes of biomarkers ( Figure 2 ). By day 8, the absolute lymphocyte counts and the PaO 2 /FiO 2 ratio were increased only among anakinra-treated patients; serum C-reactive protein (CRP) was decreased in . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1 0 both groups. By day 4, circulating ferritin was decreased only among anakinratreated patients. Notably, on the same day 4 circulating IL-6 and soluble urokinase plasminogen activator receptor (suPAR) were increased among tocilizumab-treated patients. Absolute HLA-DR receptors and the MFI of HLA-DR on circulating CD14monocytes were also increased among tocilizumab-treated patients.

Analysis of cytokine production capacity by PBMCs showed that the restoration of the cytokine potential capacity by PBMCs by day 4 was associated with better clinical outcome. More precisely, PBMCs of patients treated with anakinra had had increased production capacity for IL-6 compared to baseline before start of treatment; this was not found with tocilizumab. It was also found that the better production capacity for IL-6 by the PBMCs of anakinra-treated patients by day 4 was associated with lower scales of severity of the 28-day 11-point WHO CPS; this was not found with tocilizumab ( Figure 3 ).

Selected concurrent comparators had the same baseline characteristics as patients enrolled in the ESCAPE trial (supplementary Table 1 ). Among these 84

comparators, seven had MAS as defined by serum ferritin more than 4,420 ng/ml; six of them died by day 28 (85.7%; 95%CIs 48.7-97.4%). Among the 14 patients with MAS treated with anakinra, nine died by day 28 (64.3%; 95%CIs 38.7-83.6%); 28day mortality was lower with anakinra treatment (P: 0.033 by the binomial test).

Investigation of the two exploratory endpoints, WHO CPS and length of hospital stay favored anakinra treatment ( Figure 4 ). More precisely, survivors by day 28 who received anakinra were distributed to less severe scales than patients who received tocilizumab; this superiority of anakinra was also shown versus the concurrent comparators ( Figure 4A ). The median time to hospital discharge was 20 days with anakinra treatment and 31 days with tocilizumab treatment ( Figure 4B ).

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1 1

The incidence of secondary infections was greater with tocilizumab treatment.

The incidence of aminotransferases increase was also lower with anakinra treatment (Table 4 and supplementary Table 2 ).

A search at the Clinicaltrials.gov repository of on-going trials for COVID-19

indicates that ESCAPE is the only trial that is aiming to deliver immunotherapy to critically ill patients following a personalized approach. The approach is based on the classification of the immune function of the host i.e. MAS or CID. Results showed superior efficacy of anakinra compared to tocilizumab for the achievement of the favorable primary endpoint i.e. decrease of SOFA score and/or increase of the respiratory ratio by the end of treatment. ESCAPE was designed in March 2020 and started in April 2020 well before the WHO CPS was introduced as an endpoint for trials of COVID-19. Exploratory application of WHO CPS showed that despite the lack of difference in mortality by day 28, the allocation of patients treated with anakinra to less severe scales was more frequent compared to tocilizumab. This is compatible with the earlier discharge of anakinra-treated patients from hospital. This superiority of anakinra was also shown versus matched comparators; anakinra provided survival benefit for patients with MAS. A mechanistic insight on the function of PBMCs shows that improvement of the capacity for cytokine production is associated with better clinical outcomes. This was only found for patients treated with anakinra.

It needs to be outscored that the findings of the ESCAPE trial should not be generalized as head-to-head comparisons between the two drugs particularly since several patients were excluded from the study as they could not be immunologically . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; patients with standard-of-care; a trend for lower mortality was shown by day 14 but not by day 28 (20) . The second trial included 161 patients who were allocated to tocilizumab and 81 patients to placebo. Tocilizumab failed to prevent the incidence of mechanical ventilation or death (hazard ratio 0.83; P: 0.63) (21) . The third trial . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. (25).

The findings of the present study suggest clinical benefit among critically ill patients when anakinra treatment is guided by either ferritin more than 4,420 ng/ml which is diagnostic of MAS or with low expression of HLA-DR on CD14-circulating monocytes and increase of aminotransferases. This benefit should be confirmed in a randomized clinical trial. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. The other authors do not report any conflict of interest.

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. PBMCs were isolated before start of treatment (day 1) and on day 4 of treatment and . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint

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Abbreviations: CPK creatinine phosphokinase . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.20.21250182 doi: medRxiv preprint