Repurposed Tocilizumab in Patients with Severe COVID-19

Published December 9, 2020, doi:10.4049/jimmunol.2000981 The Journal of Immunology

Jianbo Tian,*,1 Ming Zhang,*,1 Meng Jin,†,1 Fengqin Zhang,‡ Qian Chu,‡ Xiaoyang Wang,* Can Chen,* Huihui Yue,‡ Li Zhang,x Ronghui Du,{ Dong Zhao,† Zhaofu Zeng,† Yang Zhao,† Kui Liu,‡ Mengmei Wang,† Ke Hu,† Xiaoping Miao,* and Huilan Zhang‡

The coronavirus disease 2019 (COVID-19) has caused a global pandemic, resulting in considerable morbidity and mortality. Tocilizumab, an inhibitor of IL-6, has been widely repurposed as a treatment of severely ill patients without robust evidence supporting its use. In this study, we aimed to systematically describe the effectiveness of treatment and prevention of the cytokine storms in COVID-19 patients with tocilizumab. In this multicentered retrospective and observational cohort study, 65 patients with COVID-19 receiving tocilizumab and 130 not receiving tocilizumab were propensity score matched at a ratio of 2:1 based on age, sex, and comorbidities from January 20, 2020 to March 18, 2020 in Wuhan, China. After adjusting for confounding, the detected risk for in-hospital death was lower in the tocilizumab group versus nontocilizumab group (hazard ratio = 0.47; 95% confidence interval = 0.25–0.90; p = 0.023). Moreover, use of tocilizumab was associated with a lower risk of acute respiratory distress syndrome (odds ratio = 0.23; 95% confidence interval = 0.11–0.45; p < 0.0001). Furthermore, patients had heightened inflammation and more dysregulated immune cells before treatment, which might aggravate disease progression. After tocilizumab administration, abnormally elevated IL-6, C-reactive protein, fibrinogen, and activated partial thromboplastin time decreased. Tocilizumab may be of value in prolonging survival in patients with severe COVID-19, which provided a novel strategy for COVID-19–induced cytokine release syndrome. Our findings could inform bedside decisions until data from randomized, controlled clinical trials become available. The Journal of Immunology, 2021, 206: 000–000.

oronavirus disease 2019 (COVID-19) has caused a global increased in the use of anti-inflammatory agents. However, cor- pandemic as a clinical syndrome caused by severe acute ticosteroids have short- and long-term adverse reactions (6), and C respiratory syndrome coronavirus 2 (SARS-CoV-2) (1). plasmapheresis or continuous renal replacement therapy either By August 18, 2020, the number of deaths had climbed to 767,158 requires specific equipment or lacks documented efficacy (7). among 21,549,706 confirmed cases in World Health Organization A better understanding of the underlying pathogenesis in CRS reports (2). According to a report, the mortality for critical cases facilitates the design of immunotherapies. IL-6 is the key molecule reached 60.5% (3). The elevated inflammatory cytokines suggest of CRS, so IL-6R antagonist may be of value in improving out- that cytokine release syndrome (CRS) plays a major role in the comes (8, 9). Tocilizumab is a recombinant humanized mono- pathology of COVID-19 (4, 5). clonal anti–IL‐6R Ab (10) that has been used worldwide in various Although the current focus has been on the development of novel rheumatic diseases and severe CAR T cell–induced CRS (11, 12). therapeutics, including antivirals and vaccines, there is still a long Given the pivotal role of IL-6 in COVID-19 and the efficacy way to go before the vaccine is officially launched on the market. of tocilizumab in CRS, tocilizumab was included for the first Before that, research is under way to repurpose medications. To time in Diagnosis and Treatment Protocol for Novel Coronavirus dampen excessive serum inflammatory mediators, interest has Pneumonia (seventh edition) sponsored by National Health

*Department of Epidemiology and Biostatistics, Key Laboratory for Environment Hospital of Wuhan University, Zhangzhidong Road No. 99, Wuhan 430060, Hubei, and Health, School of Public Health, Tongji Medical College, Huazhong University China (K.H.), Department of Epidemiology and Biostatistics, Key Laboratory for of Sciences and Technology, Wuhan 430030, China; †Department of Respiratory and Environment and Health, School of Public Health, Tongji Medical College, Huaz- Critical Care Medicine, Renmin Hospital of Wuhan University, Wuhan 430060, hong University of Sciences and Technology, Hang Kong Road No. 13, Han Kou China; ‡Department of Respiratory and Critical Care Medicine, Tongji Hospital, District, Wuhan 430030, Hubei, China (X.M.), or Department of Respiratory and Tongji Medical College, Huazhong University of Science and Technology, Wuhan Critical Care Medicine, Tongji Hospital, Tongji Medical College, Huazhong Uni- 430030, China; xDepartment of Oncology, Tongji Hospital, Tongji Medical College, versity of Science and Technology, Jie Fang Road, Han Kou District, Wuhan Huazhong University of Science and Technology, Wuhan 430030, China; and {De- 430030, Hubei, China (H.Z.). E-mail addresses: [email protected] (K.H.), partment of Respiratory and Critical Care Medicine, Wuhan Pulmonary Hospital, [email protected] (X.M.), or [email protected] (H.Z.) Wuhan 430030, China The online version of this article contains supplemental material. 1J.T., M.Z., and M.J. contributed equally to this work. Abbreviations used in this article: ALT, alanine transaminase; APTT, activated partial ORCIDs: 0000-0002-4641-0262 (J.T.); 0000-0002-8433-2402 (L.Z.); 0000-0001- thromboplastin time; ARDS, acute respiratory distress syndrome; AST, aspartate 9862-7239 (K.H.); 0000-0002-6818-9722 (X.M.); 0000-0002-2366-7321 (H.Z.). aminotransferase; CI, conﬁdence interval; COVID-19, coronavirus disease 2019; CRP, C-reactive protein; CRS, cytokine release syndrome; CT, computed tomogra- Received for publication August 26, 2020. Accepted for publication November 20, phy; DIC, disseminated intravascular coagulation; HR, hazard ratio; hs-cTnI, high- 2020. sensitivity cardiac troponin I; ICU, intensive care unit; IQR, interquartile range; OR, This work was supported by the SARS-CoV-2 Pneumonia Emergency Technology odds ratio; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Public Relations Project of Tongji Medical College, Huazhong University of Science This article is distributed under The American Association of Immunologists, Inc., and Technology (Grant 2020kfyXGYJ043) and the National Key Research and De- Reuse Terms and Conditions for Author Choice articles. velopment Plan for the Emergency Management of Novel Coronavirus Pneumonia, China (Grant 2020YFC0845100). Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 Address correspondence and reprint requests to Prof. Ke Hu, Prof. Xiaoping Miao, or Prof. Huilan Zhang, Department of Respiratory and Critical Care Medicine, Renmin

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000981 2 TOCILIZUMAB IN PATIENTS WITH SEVERE COVID-19

Commission of the People’s Republic of China. However, because damage was diagnosed by progressively elevated levels of alanine trans- of the small sample sizes of previous studies, there are insufficient aminase (ALT), aspartate aminotransferase (AST), or bilirubin. If the data to support the efficacy of the tocilizumab in patients with serum levels of cardiac biomarker high-sensitivity cardiac troponin I (hs- cTnI) were above the 99th percentile upper reference limit or new ab- COVID-19. To further provide a therapeutic strategy for this fatal normalities were shown in electrocardiography and echocardiography, the disease, ultimately curbing the rising fatality rate of COVID-19, acute cardiac injury was defined (19). this multicenter, retrospective, observational study presents the Safety outcomes included adverse events that occurred during treatment. clinical details of patients receiving tocilizumab from three hos- Adverse events were classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. pitals in Wuhan, China. The onset of COVID-19 was defined as the time point when the symptoms were first noticed. Patients receiving tocilizumab during hospitalization were Materials and Methods classified as the tocilizumab group. Patients who did not receive tocilizumab Study design and participants during hospitalization were classified as the nontocilizumab group. The criteria for discharge were an absence of fever for at least 3 d, substantial This multicenter, retrospective, cohort study was conducted in three hos- improvement in respiratory symptoms, and both lungs from chest CTand two pitals in Wuhan, China that were designated centers for COVID-19 throat-swab samples negative for SARS-CoV-2 RNA obtained at least 24 h treatment: Tongji Hospital, Wuhan Pulmonary Hospital, and Renmin apart (14). In this study, there were no cases lost to follow-up attributed to Hospital of Wuhan University (the east campus). We used the following standardized government management and close tracking for the COVID-19 inclusion and exclusion criteria to determine the study cohort. The inclu- pandemic. The illness severity of COVID-19 was defined according to the sion criteria included all adult patients (aged $18 y old, 5235 patients) criteria defined by the Diagnosis and Treatment Protocol for Novel Coro- with COVID-19, who were admitted to the above-mentioned hospitals in navirus Pneumonia (sixth interim edition) sponsored by National Health Hubei, China from January 20, 2020 to March 18, 2020. Eligibility criteria Commission of the People’s Republic of China. for tocilizumab administration were as follows: a diagnosis of COVID-19 confirmed upon RT-PCR positivity for SARS-CoV-2; patients with ex- Statistical analysis tensive lung lesions; severe cases who also show an increased level of IL-6 Continuous variables were presented as median and interquartile range in laboratory testing. Exclusion criteria were as follows: incomplete (IQR) or mean and SD. Categorical variables were expressed as a number medical records (e.g., transfer to any other hospital), evidence of con- (percentage). Differences between the tocilizumab group and the non- comitant bacterial infection, and pregnancy. tocilizumab group were compared. For continuous variables, a Student t test After quality control in clinical data, 65 patients using tocilizumab was used for normal distributed data and a Mann–Whitney U nonpara- (B2084B21; Roche Pharma) were ultimately enrolled, and a total of 130 metric test was used for nonnormal distributed continuous data. The patients not taking tocilizumab were statistically matched by propensity Pearson x2 test, Pearson x2 test with Yates continuity correction, or Fisher score matching (13) at a ratio of 2:1, based on age, sex, and comorbidities. exact test were applied for categorical variables. Time to events (discharge The comorbidities, including hypertension, diabetes, tumor, coronary heart or death) were defined as the time from hospital admission to events. A disease, chronic obstructive pulmonary disease, cerebral infarction, liver Cox proportional hazards model was fitted for time to death, controlling cirrhosis, hepatitis, and tuberculosis, were used as matching factors in this for treatment group and potential confounders, including age, gender, and study. These comorbidities have been reported to be risk factors for se- comorbidities. Univariable and multivariable logistic regression models verity or death of COVID-19 (14, 15). The date of final follow-up was were employed to estimate odds ratios (ORs) and 95% confidence intervals April 14, 2020. (CIs), adjusting for age, sex, and comorbidities. A two-sided p , 0.05 was For diagnosis, patients were assessed for eligibility on the basis of a considered statistically significant. All statistical analyses were performed positive RT-PCR assay (Shanghai Zhijian Biotechnology or Sansure Bio- using R (3.6.0) or SPSS Statistics (22.0). tech) for SARS-CoV-2 in a respiratory tract sample tested by the local Center for Disease Control or by a designated diagnostic laboratory. Results This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology This study cohort included 195 COVID-19 patients who were ad- (TJ-C20200108) and granted a waiver of informed consent from study mitted to Tongji Hospital, Wuhan Pulmonary Hospital, and Renmin participants. Hospital of Wuhan University (the east campus) during January 20, Data collection 2020 through March 18, 2020. Among these patients with COVID-19, 65 were classified as the tocilizumab group (71.0 y old [IQR 63.0– Epidemiological, clinical, radiological, laboratory, clinical treatments, and 75.0]; 73.85% men), and 130 patients were classified as the non- clinical outcomes data of all patients with laboratory-confirmed SARS- CoV-2 were obtained with data collection forms from electronic medical tocilizumab group (67.5 y old [IQR 61.0–75.0]; 63.08% men) records of Tongji Hospital, Wuhan Pulmonary Hospital, and Renmin according to the medication used during the hospitalization. Hospital of Wuhan University (the east campus). The admission and in- The characteristics of the tocilizumab group versus the non- hospital data of these patients were collected, reviewed, and verified tocilizumab group on admission were provided in Supplemental by a trained team of physicians to guarantee the accuracy of the data Table I and Table I. In terms of age, gender, comorbidities, extraction procedures. Any missing or uncertain records were collected and clarified through communication with involved health care pro- symptoms, and demographic characteristics (CT) of these patients, viders and their families. The detailed and standardized information of no significant differences were observed between these two demographic data, comorbidities, initial symptoms and vital signs were groups. Compared with the nontocilizumab group, the tocilizumab recorded or diagnosed at hospital admission. The chest computed to- group had higher level of D-dimer and urea. In the tocilizumab mography (CT) was recorded at hospital admission and after treatment. The complications, treatments, clinical outcomes, and hospital length of group, tocilizumab (B2084B21; Roche Pharma) was initiated at a stay were monitored until April 14, 2020, the final data of follow-up. median of 15 d (IQR 4.0–21.5) following admission. Tocilizumab Laboratory examinations, including blood routine, immune cells sub- was administered at a dose of 4–8 mg/kg body weight, and the sets, inflammatory or infection-related cytokines and biomarkers, and recommended dose was 400 mg through an i.v. drip up to a cardiac/renal/liver/coagulation function tests, were detected on the last maximum of 800 mg. Dilution was to 100 ml with 0.9% normal diagnosed date from laboratory information system. saline, and the infusion time was more than 1 h. In case of fever Outcomes within 12 h, an additional dose was given, and the cumulative dose The primary end point was in-hospital death. The main secondary outcomes could not be given more than two times. A third infusion, 24 h were total days in the intensive care unit (ICU), the time from admission to apart from the second, was optional, based on clinical response. discharge, and complications during hospitalization. Acute respiratory Additional information about dosing and administration appears in distress syndrome (ARDS) was defined according to the Berlin definition Supplemental Table II. In terms of other in-hospital treatment, (16). Disseminated intravascular coagulation (DIC) was diagnosed by increased levels of platelets, clotting factors, and other blood components there were no significant differences in both groups, suggesting (17). Acute renal injury was identified according to the Kidney Disease: that the different clinical outcomes were mainly triggered by the Improving Global Outcomes clinical practice guidelines (18). Acute liver tocilizumab treatment (Supplemental Table III). The Journal of Immunology 3

Table I. Characteristics of the overall population at baseline

Indicators Total Tocilizumab Nontocilizumab p Value Characteristics on admission Age (n = 195) in years 69.00 (62.00–75.00) 71.00 (63.00–75.00) 67.50 (61.00–75.00) 0.378 Male (n = 195) 130 (66.67%) 48 (73.85%) 82 (63.08%) 0.133 Female (n = 195) 65 (33.33%) 17 (26.15%) 48 (36.92%) Systolic pressure (n = 193) in mm Hg 131.26 6 18.15 134.40 6 18.91 129.68 6 17.62 0.087 Diastolic pressure (n = 193) in mm Hg 79.91 6 12.09 82.74 6 11.86 78.50 6 12.00 0.096 Heart rate (n = 194) 93.00 (82.75–103.00) 90.00 (79.25–101.00) 94.50 (82.75–108.00) 0.075 Respiratory rate (n = 193) 21.00 (20.00–28.00) 22.00 (20.00–29.00) 21.00 (20.00–26.25) 0.758 Temperature (n = 191) in ˚C 36.80 (36.30–37.50) 36.70 (36.30–37.05) 36.80 (36.30–37.80) 0.136 Laboratory examination on admission Inflammatory cytokines and biomarkers IL-6 (n = 195) in pg/ml 41.02 (21.31–82.43) 31.95 (18.83–74.24) 44.18 (23.81–84.72) 0.091 IL-8 (n = 180) in pg/ml 20.15 (11.15–39.8) 18.85 (7.75–37.93) 20.25 (12.58–40.08) 0.195 IL-10 (n = 180) in pg/ml 7.10 (5.00–11.63) 6.35 (5.00–9.50) 7.65 (5.00–12.20) 0.082 IL-1b (n = 180) in pg/ml 7.88 6 9.13 6.65 6 4.61 8.36 6 10.34 0.128 IL-2R (n = 179) in U/ml 835.00 (565.00–1234.0) 715.00 (468.5–1020.5) 887.50 (588.75–1331) 0.075 hs-CRP (n = 192) in mg/l 68.10 (34.58–133.60) 60.85 (23.35–139.23) 72.75 (38.63–124.10) 0.220 PCT (n = 171) in ng/ml 0.14 (0.06–0.41) 0.14 (0.07–0.36) 0.13 (0.06–0.42) 0.685 Lymphocytes count per ml Lymphocytes (n = 194) 3109/l 0.75 (0.55–1.06) 0.65 (0.47–1.06) 0.78 (0.61–1.05) 0.104 CD4+T cell (n = 64) (/ml) 243.00 (93.50–348.50) 264.00 (105.50–311.28) 217.00 (81.99–392.25) 0.731 CD8+T cell (n = 64) (/ml) 117.00 (43.27–282.00) 117.00 (56.27–255.50) 121.50 (37.75–308.25) 0.819 CD3+T cell (n = 64) (/ml) 423.00 (233.00–799.00) 379.00 (225.00–554.72) 438.50 (263.50–819.00) 0.366 CD32CD16+CD56+NK cell (n = 55) (/ml) 86.00 (38.00–143.00) 68.00 (44.00–127.00) 93.50 (27.00–150.25) 0.966 T cell+B cell+NK cells (n = 55) (/ml) 774.59 6 478.54 663.92 6 426.51 825.96 6 499.78 0.294 CD19+B cells (n = 55) (/ml) 101.00 (53.00–163.00) 75.00 (59.00–118.00) 122.50 (49.50–164.50) 0.492 Blood routine Neutrophils (n = 194) (3109/l) 6.20 6 4.26 6.57 6 3.68 6.04 6 4.50 0.388 Eosinophils (n = 194) (3109/l) 0.01 (0–0.04) 0.015 (0–0.055) 0.01 (0–0.03) 0.455 Basophils (n = 194) (3109/l) 0.01 (0.01–0.02) 0.01 (0.01–0.02) 0.01 (0.01–0.02) 0.774 Monocytes (n = 194) (3109/l) 0.43 (0.28–0.57) 0.41 (0.29–0.55) 0.45 (0.28–0.57) 0.345 Organ damage indices D-dimer (n = 179), mg/ml 1.61 (0.75–4.53) 2.57 (0.96–10.69) 1.38 (0.70–3.53) 0.046a APTT (n = 180), s 42.00 (36.9–47.13) 41.20 (35.90–49.80) 42.50 (37.70–46.70) 0.881 PT (n = 181), s 14.50 (13.68–15.7) 14.30 (13.73–15.30) 14.50 (13.60–15.80) 0.456 Fbg (n = 179), g/l 5.38 6 1.63 5.41 6 1.74 5.36 6 1.59 0.881 ALT (n = 195), U/l 27.00 (17.00–40.25) 30.00 (21.25–46.25) 24.00 (16.00–39.00) 0.078 AST (U/l, n = 195) 36.00 (25.00–53.25) 36.00 (26.00–54.75) 36.00 (25.00–51.25) 0.503 NT-proBNP (n = 163), pg/ml 368 (112.25–1013.75) 480.00 (145.00–888.00) 345.00 (99.00–1227.00) 0.930 CK (n = 142), U/l 108.50 (46.25–208.00) 80.50 (47.75–181.50) 123.00 (46.00–224.5) 0.523 CK-MB (n = 135), U/l 1.20 (0.63–2.48) 1.30 (0.85–2.30) 1.10 (0.60–2.50) 0.298 hs-cTnI (n = 163), pg/ml 10.35 (4.2–30.83) 8.00 (4.95–23.50) 11.40 (3.75–34.55) 0.868 Sixty-five patients using tocilizumab and 130 patients not taking tocilizumab were compared. Epidemiological and laboratory data on admission of all patients with laboratory-confirmed SARS-CoV-2 were obtained with data collection forms from electronic medical records of Tongji Hospital, Wuhan Pulmonary Hospital, and Renmin Hospital of Wuhan University (the east campus). Continuous variables were described as median and IQR or mean and SD, and differences were assessed using analysis of Student t test or Mann–Whitney U test. Categorical variables were expressed as number (percentage), and differences between groups were assessed using Pearson x2 test, Fisher exact test, or the continuity correction x2 tests. aData shown are significant at p , 0.05. Abbreviations used in this table: CD, cluster of differentiation; CK, creatine kinase; CK-MB, creatine kinase–myocardial band; Fbg, fibrinogen; hs-CRP, hypersensitive CRP; NT-proBNP, N-terminal brain natriuretic propeptide; PCT, procalcitonin; PT, plasma prothrombin time.

Primary outcomes SARS-CoV-2 infection can arouse both pulmonary and multi- During a follow-up duration, 56 died out of the 195 patients (28.72%) system inflammation, leading to critical complications. In this with COVID-19. The risk of in-hospital proportion of death was lower study, we observed that the incidence of ARDS (36.92 versus , in tocilizumab group versus nontocilizumab group (21.54% [14/65] 70.77%; p 0.0001) was lower in the tocilizumab group than in versus 32.31% [42/130]; p = 0.12) (Table II). In the Cox proportional the nontocilizumab group (Table II). In the multivariable logistic hazard model, after adjusting for age, gender, and comorbidities, use regression models, use of tocilizumab was associated with lower of tocilizumab was associated with lower in-hospital proportion of risk of ARDS (OR = 0.23; 95% CI = 0.11–0.45; p , 0.0001) death (hazard ratio [HR] = 0.47; 95% CI = 0.25–0.90; p =0.023) (Table III). These data suggest that IL-6 may be a potential ac- (Fig. 1). In our study, the adjusted HR for an association of known tionable target cytokine to treat COVID-19–related ARDS and variables for in-hospital proportion of death due to COVID-19 were that tocilizumab is effective in reducing the incidence of ARDS. 1.82 (95% CI = 1.01–3.27; p = 0.047) for hypertension and 3.25 Laboratory test results (95% CI = 1.27–8.30; p = 0.014) for tuberculosis. Considering that CRS refers to an uncontrolled and overwhelming Secondary outcomes release of proinflammatory mediators by an overly activated im- Total days in the ICU in the tocilizumab group are shorter than mune system, we analyzed the difference between the tocilizumab those in the nontocilizumab group (10.00 versus 12.00 d; p = 0.27), group and the nontocilizumab group based on the levels of in- and the duration from admission to hospital discharge was sig- flammatory cytokines and immune cells subsets after treatment nificantly longer (40.00 versus 26.50 d; p = 0.001) (Table II). (Table II). Analysis revealed that the infection-related biomarkers, 4 TOCILIZUMAB IN PATIENTS WITH SEVERE COVID-19

Table II. Patient characteristics in tocilizumab group and nontocilizumab group after treatment

Indicators Total Tocilizumab Nontocilizumab p Value Outcome of disease ICU admission (n = 195) 110 (56.41%) 40 (61.54%) 70 (53.85%) 0.307 Total days in ICU (n = 110) 11.00 (7.00–19.00) 10.00 (3.50–18.75) 12.00 (8.00–19.00) 0.271 Days from admission to discharge (n = 29.00 (22.00–41.00) 40.00 (25.00–34.00) 26.50 (22.00–34.00) 0.001a 139) Nonsurvival (n = 195) 56 (28.72%) 14 (21.54%) 42 (32.31%) 0.117 Survival (n = 195) 139 (71.28%) 51 (78.46%) 88 (67.69%) Inflammatory cytokines and biomarkers IL-6 (n = 195) in pg/ml 70.44 (31.62–450.85) 104.00 (38.18–515.40) 52.01 (29.97–440.78) 0.123 IL-8 (n = 180) in pg/l 19.10 (10.98–77.28) 16.45 (9.35–42.35) 21.40 (11.30–123.50) 0.201 IL-10 (n = 180) in pg/ml 6.30 (5.00–17.43) 5.00 (5.00–11.98) 7.35 (5.00–18.40) 0.013a IL-1b (n = 180) in pg/ml 5.00 (5.00–8.45) 5.00 (5.00–8.48) 5.00 (5.00–8.45) 0.797 IL-2R (n = 179) in U/ml 790.00 (548.50–1300.50) 708.00 (507.25–1120.75) 839.00 (566.50–1324.00) 0.178 hs-CRP (n = 192) in mg/l 14.35 (1.65–83.38) 1.52 (0.40–21.63) 22.55 (4.68–111.50) ,0.0001a PCT (n = 171) in ng/ml 0.09 (0.05–0.82) 0.06 (0.04–0.60) 0.11 (0.05–0.90) 0.235 Lymphocytes Lymphocytes count (n = 194) (3109/l) 0.96 (0.54–1.47) 0.96 (0.51–1.27) 0.98 (0.54–1.54) 0.689 CD4+T cell count (n = 64) (/ml) 339.50 (237.25–495.75) 395.00 (276.51–644.00) 305.00 (174.50–429.00) 0.064 CD8+T cell count (n = 64) (/ml) 151.00 (53.75–316.50) 256.98 (149.50–356.26) 78.00 (39.25–256.75) 0.003a CD3+T cell count (n = 64) (/ml) 422.03 (216.50–757.00) 608.29 (447.80–954.25) 300.00 (117.50–531.50) 0.001a CD32CD16+ CD56+NK cell (n = 55) (/ml) 74.00 (22.50–139.50) 112.00 (66.00–158.00) 45.50 (16.75–127.50) 0.053 T cell+B cell+NK cell (n = 55) (/ml) 729.00 (451.00–1029.00) 834.00 (587.00–978.00) 665.50 (356.00–1052.50) 0.219 CD19+B cell (n = 55) (/ml) 117.00 (46.50–165.50) 113.00 (64.00–144.00) 119.50 (45.25–170.00) 0.689 Blood routine Neutrophils (n = 194) (3109/l) 5.23 (3.33–9.20) 5.93 (3.48–9.13) 4.67 (3.24–9.18) 0.521 Eosinophils (n = 194) (3109/l) 0.07 (0.01–0.18) 0.06 (0.00–0.14) 0.08 (0.02–0.18) 0.265 Basophils (n = 194) (3109/l) 0.02 (0.01–0.04) 0.02 (0.01–0.04) 0.02 (0.01–0.04) 0.131 Monocytes (n = 194) (3109/l) 0.48 (0.31–0.68) 0.46 (0.27–0.69) 0.48 (0.34–0.68) 0.847 Organ damage indices D-Dimer (n = 179) (mg/ml) 1.55 (0.63–3.33) 1.53 (0.73–2.95) 1.69 (0.62–3.88) 0.446 OD-Dimer (n = 179) (mg/ml) 0.00 (21.33–0.86) 20.46 (26.72 to 1.31) 0.00 (2078–0.71) 0.030a APTT (n = 180) (s) 40.75 (35.08–47.05) 36.80 (34.70–42.10) 42.30 (37.10–48.35) 0.006a PT (n = 181) (s) 14.20 (13.30–16.20) 13.90 (13.20–15.70) 14.35 (13.50–16.25) 0.050 Fbg (n = 179) (g/l) 4.16 (2.96–5.21) 2.57 (2.02–3.72) 4.71 (3.71–5.67) ,0.0001a ALT (n = 195) (U/l) 23.00 (15.00–36.00) 24.00 (17.00–37.00) 22.00 (15.00–36.00) 0.570 AST (n = 195) (U/l) 24.00 (19.00–40.00) 22.00 (19.00–40.00) 25.00 (19.00–40.75) 0.452 NT-proBNP (n = 163) (pg/ml) 346.50 (99.50–3660.00) 285.50 (107.75–3418.25) 446.00 (92.50–3683.00) 0.884 CK (n = 142) (U/l) 47.00 (30.00–108.25) 40.00 (30.00–59.75) 53.00 (29.25–179.75) 0.051 CK-MB (n = 135) (U/l) 1.10 (0.60–3.45) 1.00 (0.60–2.80) 1.10 (0.60–4.95) 0.925 hs-cTnI (n = 163) (mg/l) 10.40 (3.15–44.00) 5.60 (2.95–50.30) 11.95 (3.50–39.05) 0.301 Sixty-five patients using tocilizumab and 130 patients not taking tocilizumab were compared. Laboratory and clinical outcomes data after treatment of all patients with laboratory-confirmed SARS-CoV-2 were obtained with data collection forms from electronic medical records of Tongji Hospital, Wuhan Pulmonary Hospital and Renmin Hospital of Wuhan University (the east campus). Continuous variables were described as median and IQR or mean and SD, and differences were assessed using analysis of Student t test or Mann–Whitney U test. Categorical variables were expressed as number (percentage), and differences between groups were assessed using Pearson x2 test, Fisher exact test, or the continuity correction x2 tests. aData shown are significant at p , 0.05. Abbreviations used in this table: CD, cluster of differentiation; CK, creatine kinase; CK-MB, creatine kinase–myocardial band; Fbg, fibrinogen; hs-CRP, hypersensitive CRP; NT-proBNP, N-terminal brain natriuretic propeptide; PCT, procalcitonin; PT, plasma prothrombin time. including IL-10 (5.00 versus 7.35 pg/ml; p = 0.013) and C-reactive tocilizumab treatment, CRP decreased from 60.85 to 1.52 mg/l protein(CRP;1.52versus22.55mg/l;p , 0.0001) were re- in tocilizumab group, and the number of immune cells was markably decreased in the tocilizumab group compared with higher than the baseline, especially in the tocilizumab group. the nontocilizumab group (Table II). Consistent with recently The difference did not reach statistical significance, but it published studies, results revealed that CRS-related cytokines, presented a statistical trend, which may be attributed to the especially CRP, were rapidly reduced in patients with severe limited sample size and short follow-up time. Noticeably, we COVID-19 after receiving tocilizumab (20, 21). In contrast, observed that CRP and T cell numbers appeared as a signifi- the count of CD3+ T cells (608.29 versus 300.00/ml; p =0.001) cant change in tocilizumab group at week 7 compared with the was significantly increased in tocilizumab group than that in control group. It is warranted to evaluate the effect of tocili- the nontocilizumab group (Table II). For further analysis of zumab in larger scale prospective cohort studies and ran- T cell subsets, we found that the count of CD4+ T cells (395.00 domized controlled trials. versus 305.00/ml; p =0.064)andCD8+ T cells (256.98 versus We also evaluated the dynamic changes of organ damage indices 78.00/ml; p = 0.003) was increased as well as the total number at final examination (Table II). Biomarkers of multiple organ of T cells (Table II). These findings indicate that COVID-19 dysfunction in the tocilizumab group were within the normal patients using tocilizumab have more improved inflammation range. After the treatment with tocilizumab, the activated coagu- and immune cell function recovery than those who did not. The lation system by IL-6 returned to normal. Activated partial dynamic change of four indices over follow-up are shown in thromboplastin time (APTT) was shorter than in the non- the Fig. 2. After the patient was admitted to the hospital, the tocilizumab group (36.80 versus 42.30 s; p = 0.006), and the de- level of CRP increased, whereas CD3+ TcellandCD8+ Tcell cline of fibrinogen (2.57 versus 4.71 g/l; p , 0.0001) was more counts declined in both groups as the disease progressed. After pronounced in tocilizumab group (Table II). The Journal of Immunology 5

FIGURE 1. Survival of COVID-19 patients during hospitalization. Kaplan–Meier curves for cumulative p value of COVID-19 mortality during follow up duration in tocilizumab or non-tocilizumab cohort among 195 patients within Cox proportional hazards model.

Adverse event the People’s Republic of China to continue tocilizumab in patients The percentages of patients with adverse reactions were similar in with severe or critical COVID-19 (14, 23). Given the observa- the two groups; there were significant differences (Supplemental tional design, whether tocilizumab could reduce overall mortality Table IV). The most common adverse event in the tocilizumab in patients with COVID-19 needs further validation in geograph- group was increasing liver enzymes (AST, 8/65 [12.31%]; ALT, ically diverse, large-scale prospective cohort studies. 9/65 [13.85%]) as previously reported (22). CRS was found to be the major cause of morbidity in patients infected with severe SARS-CoV and MERS-CoV (8). Elevated serum concentrations of the IL-6 are hallmarks of severe MERS- Discussion CoV infections (24). Elevated serum CRP, a protein whose In this multicenter retrospective study involving a modest sample of expression is driven by IL-6, is also a biomarker of severe consecutive patients who had been hospitalized with COVID-19, b-coronavirus infection. Recent studies have also reported that the risk of in-hospital proportion of death was significantly hypercytokinemia is associated with a higher proportion of death lower among patients who received tocilizumab than among those in patients with severe COVID-19. IL-6 is an excellent biomarker who did not. It revealed that tocilizumab could prolong survival of severity and a prognostic indicator for most of diseases pre- among patients with COVID-19, specifically among hospitalized senting with a cytokine storm and is expressed longer than TNF-a COVID-19 patients. Clinical data showed that tocilizumab treat- and IL-1 (25). Although IL-6 is regulated strictly through tran- ment weakens the inflammatory response while sustaining immune scription and posttranscription mechanisms, dysregulated, persis- responses in posttreatment COVID-19 patients, suggesting that tent production of IL-6 plays a pathological role in tissue hypoxia, tocilizumab could be an efficient therapeutic for the treatment of hypotension, DIC, and multiple organ dysfunction (26–29). COVID-19. Although it is plausible that unmeasured confounding Mechanistically, IL-6 is essential for the generation of Th17 cells factors may have contributed to the observed protective associa- in the dendritic cell–T cell interaction (30). The excessive IL-6 tion, these data suggested that in-hospital use of tocilizumab was may explain the overly activated Th17 cells observed in COVID- not associated with increased proportion of death in COVID-19. 19 patients (31). A high level of IL-6 is associated with poor These findings provide clinical evidence in support of recently outcome in the setting of COVID-19 pneumonia (32). Accumu- published guidance statements by National Health Commission of lating evidence evoked the use of tocilizumab in COVID-19

Table III. HR for outcomes in tocilizumab group versus nontocilizumab group

Univariable Logistic Regression Multivariable Logistic Regression

Complications OR (95% CI) p Value OR (95% CI) p Value ARDS 0.24 (0.13–0.45) ,0.0001a 0.23 (0.11–0.45) ,0.0001a DIC 0.28 (0.03–2.28) 0.231 0.26 (0.03–2.37) 0.232 Acute renal injury 0.78 (0.39–1.57) 0.49 0.69 (0.31–1.51) 0.35 Acute liver injury 1.63 (0.78–3.32) 0.203 1.75 (0.78–3.92) 0.175 Acute cardiac injury 1.14 (0.62–2.09) 0.679 0.98 (0.49–1.98) 0.960 Sixty-five patients using tocilizumab and 130 patients not taking tocilizumab were compared. Complication data after treatment of all patients with laboratory-confirmed SARS-CoV-2 were obtained with data collection forms from electronic medical records of Tongji Hospital, Wuhan Pulmonary Hospital, and Renmin Hospital of Wuhan University (the east campus). The multivariable logistic regression model was adjusted for age, sex, and comorbidities. ORs and 95% CIs were calculated by univariable logistic regression models. aData shown are significant at p , 0.05. 6 TOCILIZUMAB IN PATIENTS WITH SEVERE COVID-19

FIGURE 2. Temporal changes in laboratory parameters in COVID-19 patients after admission. The temporal changes of hypersensitive CRP (hs-CRP) (A), CD3+ T cell (B), CD8+ T cell (C), and lymphocyte (D) in tocilizumab group and nontocilizumab group. These parameters were measured at 1, 2, 3, 4, 5, 6, and 7 wk after admission. The green dotted line represents the lower limit of normal, and the red dotted line represents the upper limit of normal. The levels of these parameters at each point were expressed as median and IQR. **p , 0.01, ***p , 0.001 were calculated by Mann–Whitney U test for last examination. infection. Tocilizumab, an IL-6R monoclonal blocking agent, was the same time, patients deteriorated with increased inflammatory approved for the treatment of rheumatoid arthritis in Japan (2008), response (inflammatory cytokines were elevated [e.g., IL-6, CRP]) Europe (2009), and the United States (2010) (4, 33–35). Mecha- and decreased immune function (immune cells decreased [e.g., nistically, tocilizumab can bind both soluble and membrane-bound CD4+ T cell, CD8+ T cell]) before tocilizumab. After the treat- IL-6R to inhibit IL-6–mediated cis- and trans-signaling (10). A ment, in addition to partially rescuing the immune dysregulation, new study found that the inhibition of IL-6 signaling by tocili- the inflammatory response was weakened to some degrees in the zumab treatment decreased PAI-1 production and improved the patients. During the treatment, no severe adverse drug reactions vascular endothelial functions in patients with severe COVID-19. were reported. Clinical symptoms of all patients improved with IL-6 trans-signaling forms a positive feedback loop and plays good prognosis after the treatment. Overall, these findings pivotal roles in endothelial cell injury and coagulopathy, thereby suggested that tocilizumab group seemed to have an improved mediating systemic inflammation and deteriorating systemic cir- survival outcome that might be explained by the blocking of culation (36). A phase II trial is ongoing in Italy (37), and a phase IL-6–associated dysregulated immune responses and inflam- III trial was approved by the Food and Drug Administration (38) matory storm response. to assess the effect of tocilizumab for severe COVID-19 pneu- Our study has several limitations. First, this retrospective study monia. More than 1 million patients worldwide have been treated population is derived from three hospitals in Hubei Province, but with tocilizumab, and clinical trials have proved that tocilizumab the effects of tocilizumab may vary among people of different races is safe for both pediatric and adult patients (22). In the mainly or geographic locations, which seems reasonable. Second, the rheumatologic indications, the tolerance of tocilizumab is gener- potential mechanisms of dysregulated inflammatory cytokines and ally good, the main adverse events are a transient decrease in immune responses were not fully explored and need to be further leukocytes, an increase in liver enzymes, and a slight increase of investigated. Third, the sample size of the study was modest and bacterial infection (22). included 65 patients who received tocilizumab. Currently, the In COVID-19 patients with elevated inflammatory cytokines, sample size enrolled in case-control study was the largest. Because postmortem pathology has revealed tissue necrosis and interstitial unmeasured biases are particular problems inherent in retrospective macrophage and monocyte infiltrations in the lung, heart, and studies, we adjusted for likely confounders, including age, sex, and gastrointestinal mucosa (22). Moreover, severe lymphopenia with comorbidities in our analysis. Despite this extensive adjustment, it hyperactivated proinflammatory T cells and decreased regulatory is still possible that some amount of unmeasured confounding T cells are commonly seen in critically ill patients, suggesting remains. Therefore, large-scale prospective cohort studies and dysregulated immune responses. In our study, 65 patients had a randomized controlled trials are needed to better understand the history of routine treatment for ∼15 days before tocilizumab efficacy of tocilizumab in COVID-19. Recently a randomized, treatment. A lower percentage of lymphocytes was found in pa- double-blind, placebo-controlled trial indicated that tocilizumab tients, and it returned to normal when tocilizumab was given. At had no significant effect on the risk of intubation or death (39). The Journal of Immunology 7

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