Extracorporeal cytokine removal in severe CAR-T cell associated cytokine release syndrome

Journal Pre-proof Extracorporeal cytokine removal in severe CAR-T cell associated cytokine release syndrome

Klaus Stahl, Bernhard M.W. Schmidt, Marius M. Hoeper, Thomas Skripuletz, Nora Möhn, Gernot Beutel, Matthias Eder, Tobias Welte, Arnold Ganser, Christine S. Falk, Christian Koenecke, Sascha David PII:

S0883-9441(19)31831-3

DOI:

https://doi.org/10.1016/j.jcrc.2020.02.010

Reference:

YJCRC 53496

To appear in:

Journal of Critical Care

Please cite this article as: K. Stahl, B.M.W. Schmidt, M.M. Hoeper, et al., Extracorporeal cytokine removal in severe CAR-T cell associated cytokine release syndrome, Journal of Critical Care(2019), https://doi.org/10.1016/j.jcrc.2020.02.010

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© 2019 Published by Elsevier.

Journal Pre-proof

Extracorporeal cytokine removal in severe CAR-T cell associated cytokine release syndrome Klaus Stahl MD1 , Bernhard MW Schmidt MD2 , Marius M Hoeper MD3 , Thomas Skripuletz MD4 , Nora Möhn MD4 , Gernot Beutel MD5 , Matthias Eder MD5 , Tobias Welte MD3 , Arnold Ganser MD5 , Christine S. Falk PhD6 , Christian Koenecke MD5 and Sascha David MD2

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Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany Department of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany

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Department of Respiratory Medicine and German Centre o f Lung Research (DZL), Hannover Medical School, Hannover, Germany Department of Neurology, Hannover Medical School, Hannover, Germany

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Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany

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Department of Hematology, Hemostasis, Oncology and Stem cell transplantation, Hannover Medical School, Hannover, Germany

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Correspondence to: Sascha David, MD

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Medical School Hannover, Department of Nephrology & Hypertension

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Carl-Neuberg-Str.1, 30625 Hannover, GERMANY (+49) 511 532-6319, [email protected]

Conflict of interest statement: KS and SD received travel grants from C ytoSorbents Corp. Otherwise, the authors declare that they have no competing interests.

Financial disclosure statement: None.

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Journal Pre-proof Email addresses of all authors : [email protected]

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[email protected]

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Journal Pre-proof A BST RA C T Purpose: Life-threatening complications of CD-19 Chimeric antigen receptor - T (CAR-T) cells such as the cytokine release syndrome (CRS)) have been reported. Treatment is limited to IL-6 blockade and steroids although global removal of elevated soluble inflammatory factors might be more effective. Methods: Clinical course of a CRS patient treated with extracorporeal cytokine

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adsorption (Cytosorb®). A panel of 48 cytokines, chemokines and endothelial markers has been analyzed longitudinally. Ex vivo stimulation of endothelial cells to visualize

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(immunocytochemistry) and quantify (ECIS, TER) endothelial barrier effects.

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Results: Following CAR-T cell application a 65 years old male developed grade 4

resuscitation).

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CRS with refractory shock (3 vasopressors) and severe capillary leakage (+37L/ 24h Treatment

included

IL-6

blockade,

methylprednisolone

and

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additionally Cytosorb hemoperfusion. While multiple soluble inflammatory factors

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were elevated and most of them decreased by more than 50% following Cytosorb,

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markers of endothelial injury increased steadily (e.g. Angpt-2/Angpt-1) leading to profound endothelial activation and leakage in ex vivo assays. Conclusion: This is the first reported use of cytokine adsorption for CRS showing efficacy in absorption of various cytokines but not endothelial growth factors. A randomized controlled trial to evaluate additional Cytosorb treatment in CRS is currently recruiting at our institution (NCT04048434).

KEYWORDS CAR-T-cell, Cytokine release syndrome, extracorporeal cytokine adsorption

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Journal Pre-proof IN TR OD U CTIO N Treatment with chimeric antigen receptor (CAR)-T cells represents a novel treatment approach for patients with B- lineage acute lymphoblastic leukemia (ALL) (1) or relapsed and refractory aggressive B-cell lymphomas (2-4). Currently, two preparations are approved: Tisagenlecleucel and Axicabtagene Ciloleucel. The cellular therapy is generated from autologous lymphocytes by lentiviral gene transfer

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leading to the expression of a CD19 recognizing surface receptor. Subsequently, these genetically modified T-cells recognize and kill all target cells carrying the respective

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target structure (5). Through this innovative strategy, a high rate of long-term

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remissions in patients with otherwise immunochemotherapy-refractory malignancies

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was achieved in pivotal studies (6). However, frequent and potentially life-threatening complications, i.e. the cytokine release syndrome (CRS), tumor lysis (TLS) and

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immune effector cell associated neurotoxicity syndrome (ICANS) have been reported

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(5). These diseases have been defined as a systemic inflammatory condition caused by a strong and widespread immune activation induced by the CAR-T cell- mediated

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immune response (7, 8). This was shown to result in the release of various proinflammatory cytokines, including IL-6 (8). In the studies investigating the use of Tisagenlecleucel, 58-77% of patients developed any grade of CRS and 22-47% required treatment at an intensive care unit (ICU) (1, 3). Severe forms of CRS can lead to vasoplegia and capillary leakage. Tocilizumab that inhibits binding of IL-6 to its receptor both in cell-bound and circulating forms (9) was recently approved by the FDA for the treatment of CRS (10, 11). However, rapidly progressive courses of CRS despite immediate administration of Tocilizumab have been repeatedly described (12, 13). Experts have recommended in such cases up to 3 additional Tocilizumab doses and administration followed by methylprednisolone in high doses (2 mg/kg) (8).

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Journal Pre-proof Given that a complex cytokine network rather IL-6 alone is involved in CRS and that IL-6 blockade with IL-6 receptor mAb cannot cross the blood brain barrier (BBB), yet many patients develop ICANS, it appears reasonable to investigate additional therapeutic strategies. Based on these theoretical considerations, we have additionally used extracorporeal cytokine adsorption (CytoSorb® ) in a patient suffering from severe CRS and analyzed the clinical course of soluble inflammatory factors including several cytokines, chemokines, endothelial and permeability regulating

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factors as well as the effect of this patients’ plasma on the induction of endothelial

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CASE DESCRIPTION

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permeability in vitro.

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A 64-year old male patient was admitted to the ICU after he had received a preparation of CD19-specific CAR-T cells (Tisagenlecleucel, CD3+ cells: 2.5804x108 ,

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vitality 83.2%) six days prior for treatment of relapsed diffuse large B-Cell lymphoma

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(DLBCL) after three lines of chemotherapy and previous autologous stem cell

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transplantation. The patient developed episodes of fever, mild hypotension and dyspnea at day 4 and was transferred to the ICU for further monitoring. He was afebrile, awake and fully orientated but did show mild signs of encephalopathy. Mean arterial pressure (MAP) was 63 mmHg with a mild tachycardia of 115 bpm and a normal peripheral O2 saturation (SpO2) of 96% while breathing ambient air and passing clear urine at 100cc/h. Laboratory evaluations were unremarkable except raised levels of C-reactive protein (215 mg/L). At this point, the patient was diagnosed with grade 2 CRS following Lee Santomasso consensus criteria (14) so that he received a single dose of Tocilizumab at the recommended dose of 8 mg/kg (600 mg). The further clinical course and initiated treatment measures are displayed in

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Journal Pre-proof Figure 1. The patient received about 5L of crystalloid fluid substitution the following 24 hours and remained clinically stable. Sudden onset of severe shock occurred 24 hours later so that, in addition to further aggressive volume expansion, high doses of norepinephrine (NE) were necessary to maintain MAP above 65 mmHg. Extended hemodynamic measurements revealed a cardiac index (CI) of 1.54 l/min/m2 mainly due to a severely reduced preload (GEDI 434 ml/m2 , SVV 35%). Somewhat surprisingly the systemic vascular resistance was found elevated (SVRI 3273

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dyn*s*cm-5 *m2 ) indicating grave underfilling and low preload due to massive capillary leakage as the primary shock mechanism rather than vasoplegia. The patient

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rapidly became anuric and developed lactic acidosis and was therefore treated with

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SLEDD using the Genius® system. Inotropic support with dobutamine was

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additionally started (although the pathophysiological problem behind the low CO was still the underfilling rather than a myocardial contractility problem). Tocilizumab was

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administered 3 more times 8 hourly. The patient was intubated and mechanically

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ventilated due to progressive encephalopathy ultimately leading to loss of

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consciousness. Given that the patient was unresponsive to repetitive IL-6 blockade, we additionally applied extracorporeal cytokine adsorption (CytoSorb® ), which was combined with the dialysis circuit. At first, the hemodynamic situation further deteriorated and the clinical course over the next 24 hours was extremely critical. NE dose increased up to 0.53 ug/kg/min and epinephrine (EPI) was additionally required (0.21 ug/kg/min) to maintain organ perfusion. The CytoSorb® dose was then intensified to 8 hourly changes. Within the next hours, shock completely reversed – vasopressor dosage could be reduced to about 1/10 of the peak dose, inotropic support was stopped completely and intensity of dialysis and CytoSorb® treatment was decreased. The patient recovered further the following days: He was extubated and

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Journal Pre-proof completely off vasopressors on day 8 and then transferred to the hemato-oncological step-down unit two days after. Unfortunately, the patient was re-admitted to the ICU with neutropenia and a triad of 1) vancomycin resistant enterococcus (VRE) sepsis, 2) secondary hemophagocytic lymphohistiocytosis (HLH) and 3) overt cytomegalovirus (CMV) reactivation. Despite targeted therapeutic measures our patient died on day 23 after CAR-T-cell

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therapy.

MA TER IAL A ND METH OD S

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Measurement of circulating cytokines and permeability factors

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Plasma of the patient was obtained before and following start of Cytosorb® treatment

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8 hourly at day 2 (0, 8, 16 and 24 hours) when the patient was severely sick (see Figure 1, marked I-IV).

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Fifty plasma proteins, i.e. cytokines, chemokines, endothelial and growth factors were

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quantified at the protein level using the Luminex-based multiplex technology as

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described previously (human BioPlex Assay #12007283) (15). Angiopoietin-1 and -2 (Angpt-1 and -2) were measured by enzyme- linked immunosorbent assays (R&D systems, Minneapolis). Cytokines, chemokines, ligands and vasoactive proteins in Tables 1 and 2 were categorized as decreased during cytokine adsorption if the 24hr concentration was at least 15% lower than baseline before treatment. All other were categorized as unchanged or increased despite cytokine adsorption.

Endothelial ex vivo stimulation

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Journal Pre-proof Human umbilical vein endothelial cell (HUVECs) were isolated from umbilical vein donors (ethical No. 1303–2012) and cultured as described before (16). In order to mimic the CRS associated vascular phenotype in confluent HUVEC monolayers, their growth medium was supplemented with 5% plasma obtained from the patient at the time points described above.

Fluorescent immunocytochemistry

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Thirty minutes after treatment with patient plasma, cells were fixed in 2.5% paraformaldehyde, permeabilized and incubated with primary antibody (VE-cadherin

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[BD Bioscience, San Diego, CA]), followed by with secondary Alexa-antibody and

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phalloidin (17). Cell-cell contacts were analyzed by the adherens junction protein VE-

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cadherin (green) whereas the cytoskeletal architecture was visualized by F-actin (red).

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Transendothelial electrical resistance (TER)

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To quantify endothelial permeability, serial TERs were recorded with an electric cell-

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substrate impedance sensing (ECIS) approach in triplicates (Ibidi, Applied BioPhysics Inc.) as described before (18).

R ESU LTS Cytokines, chemokines, growth factors and markers of endothelial activation at admission and during the initial 24 hrs of CRS are summarized in Table 1 and 2. After 24 hrs of Cytosorb treatment, a wide range of pro-inflammatory cytokines were lowered by more than 50% compared to the concentration before start of Cytosorb treatment and the peak concentrations reached within the CRS course (Table 1). Importantly, IL-6, IFN-amma, TNF-, IL-1 and IL-1 - associated chemokines

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Journal Pre-proof (CCL27/CTACK and CXCL1/GRO-a) were decreased substantially during cytokine adsorption. A number of proteins first increased further during cytokine adsorption and could only be reduced after the dose was increased meaning that the adsorbers were changed in shorter intervals (8 hourly). Among those were IL-2, IL-4, IL-10 and Interferon-alfa2 as well as the chemokines CXCL9/MIG, CXCL10/IP10 and the growth factor G-CSF (Table 1). Certain cytokines and chemokines remained

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unchanged or even increased further during the CRS course despite cytokine adsorption (Table 2). Among these were IL1-RA, IL-18, the chemokine

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CXCL8/IL_8, CCL3-5 as well as the growth factors S-CGF, PDGF, MIF and HGF.

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Strikingly, markers of endothelial injury and activation such as the adhesion

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molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion

adsorption (Table 2).

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molecule-1 (VCAM-1) increased substantially and steadily independent of cytokine

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Angpt-1, responsible for endothelial survival and maintenance of barrier function

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mediated through Tie-2 activation, was reduced from the beginning compared to healthy controls (data not show) and declined further by about 85% during CRS course. At the same time, the antagonistic counterpart Angpt-2 was found increased compared to healthy controls and could be reduced only slightly by 25% in the further CRS course. Consequentially, the ratio between Angpt-1 and /-2 was severely increased up to 50 times higher than in healthy controls and worsened further during the course of CRS (Table 1 and 2). When the direct effect of the patient’s plasma on endothelial cells was visualized by fluorescent immunocytochemistry, formation of focal adhesions, actin stress fibers, and multiple paracellular gaps could be noticed (Figure 2A). In fact, these changes,

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Journal Pre-proof characteristic of endothelial activation and injury, progressed during the further course of CRS. Consistently, endothelial cells stimulated with patient’s plasma showed an immediate drop in transendothelial electrical resistance (TER) in real-time using ECIS measurements – the equivalence of the clinically observed capillary leakage syndrome. These changes were still reversible in samples at 0 and 8hrs but no longer reversible with the later samples at 16 and 24 hrs indicating progressive endothelial

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leakage within the time course of CRS (Figure 2B).

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DISCUSSION

The patient presented with progressive shock due to CRS. Despite treatment with

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repetitive doses of Tocilizumab, methylprednisolone and massive volume substitution

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the clinical course rapidly deteriorated requiring vasopressor and inotropic support at exceedingly high doses. With the patient being unresponsive to above treatments, we

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decided to employ an extracorporeal removal strategy by continuous cytokine hemoadsorption. In case reports and smaller studies on various conditions with increased systemic inflammation, e.g. septic shock, pancreatitis etc., additive treatment with CytoSorb® showed rapid hemodynamic stabilization (19-21). To our knowledge this is the first CRS patient that has been treated with an extracorporeal cytokine adsorption strategy. We followed this approach based on a following theoretical rationale: 1) Following Tocilizumab administration, blood levels of IL-6 may still continue to rise as endocytosis of IL-6 is the most important elimination mechanism (22).

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Journal Pre-proof Interestingly, an increase in plasma IL-6 after Tocilizumab administration was also observed in our patient (Table 1, 0 vs. 8 hours). 2) IL-6 can easily cross the blood brain barrier (BBB) and it is well-known for its neurotoxic effects (23). In contrast, Tocilizumab is not able to cross the BBB, preventing pharmacological action in the central nervous system (24). It has therefore been proposed that the administration of Tocilizumab may transiently even worsen ICANS (8). In fact, our patient needed to be intubated primarily due to severe

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encephalopathy following administration of 3 Tocilizumab doses.

3) Considering a situation with reduced clearance and excessive further release of IL-

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6 (and many other cytokines) into the circulation and into compartments that are not

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accessible to Tocilizumab, it appears reasonable to employ techniques aiming to

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remove excessive cytokines from the circulation rather than just blocking its biological function.

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4) Equally important, IL-6 is by no means the only pro- inflammatory cytokine that

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appears to be involved in the pathogenesis of CRS. Increased plasma concentrations

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for many pro- inflammatory molecules have been reported: IFNγ, TNFα, IL-1, IL-2, IL-5, IL-8, IL-10, IL-15, GM-CSF and CCL2/MCP-1 (7, 10, 25, 26). Interestingly, it has been shown that the increase of CCL2/MCP-1 in the blood of patients after CART cell therapy is more specific for the prediction of a severe CRS course than IL-6 (7). Especially for IL-1 and IL-1 receptor signaling, a critical role in the development of generalized vascular dysfunction in CRS was recently described in an animal model (27). CytoSorb® is a commercially available hemoadsorption device that unselectively removes proteins including cytokines from the blood plasma by adsorption onto a biocompatible porous polymer sorbent with a large surface area (approximately 40,000 m2 ) (28). In in vitro and in vivo experiments, CytoSorb® has been shown to

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Journal Pre-proof remove a wide range of pro- inflammatory cytokines ranging in size from 5 to 60 kDa, including but not restricted to IL-6 (29-31). We observed that additive cytokine adsorption was able to modulate a wider spectrum of pro- inflammatory mediators of CRS beyond IL-6. In fact, previously mentioned additional signature cytokines of CRS such as IFN-γ, TNF-α, IL-1, IL-5 and CCL2/MCP1 could all be reduced substantially during cytokine adsorption. With the use of the first CytoSorb ® adsorber over 16 hours, many cytokines such as

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IL-2, IL-4, IFN-a, CXCL9/ and -10 as well as CCL3-5 were unchanged. It is possible that saturation of the adsorber by excessive cytokine levels and continuous de novo

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generation did occur and the “lifespan” (or adsorption capacity) was therefore

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significantly reduced. In line with this hypothesis, many cytokines decreased

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following an exchange of the adsorber. If a higher adsorption dose every 8 hours, such as employed later in this patient, might have contributed to clinical stabilization

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remains speculative. Given the limitations of an uncontrolled case report it is

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important to us to highlight that one can only speculate how much of the change in

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cytokines was indeed attributable to Cytosorb ® or simply reflected the natural disease course.

The most striking clinical manifestation of CRS in this case was not vasoplegia but extreme vascular leakage indicated by severely reduced preload despite excessive volume expansion. Markers of endothelial injury such as the soluble adhesion molecules ICAM-1 and VCAM-1 increased steadily within the clinical course and we could

later

demonstrate

severe

endothelial

activation

by

fluorescent

immunocytochemistry and real time TER and leakage ex-vivo. Recent evidence points to an important role of endothelial permeability factors such as the ratio between angiopoietin-2/-1, which increases in proportion to the severity of

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Journal Pre-proof CRS in the blood of patients and contributes to the clinically significant permeability disorder (7).

CONCLUSI ONS In conclusion, our findings suggest the possibility that removal of excessive circulating cytokines rather than pharmacological blockade of a single key cytokine alone might be a more effective treatment strategy for severe CRS. Therefore, we

treatment

with CytoSorb®

hemoadsorption

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have initiated a prospective randomized controlled study investigating extracorporeal in patients

with severe CRS

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(clinicaltrials.gov; NCT04048434), which is now recruiting at our institution.

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LIST O F ABBREVI ATIO NS Angpt = Angiopoietin

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ADAMTS13 = a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 BBB = blood brain barrier

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CAR-T-cell = chimeric antigen receptor – T-cell

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CCL = CC motif chemokine CMV = cytomegalovirus

CRS = cytokine release syndrome

CTACK = Cutaneous T-cell attracting chemokine /CCL CVVHD = continuous veno-venous hemodialysis CXCL = C-X-C motif chemokine Dobu = dobutamine ECIS = electric cell-substrate impedance sensing Epi = epinephrine FGF = Fibroblast growth factor Genius® = renal replacement therapy using the Genius hemodialysis system G-CSF = Granulocyte colony-stimulating factor

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Journal Pre-proof HGF = hepatocyte growth factor HLH = hemophagocytic lymphohistiocytosis Hu-SCF = Human stem cell factor HUVECs = human umbilical vein endothelial cells ICAM-1 = Intercellular adhesion molecule-1 ICANS = immune effector cell associated neurotoxicity syndrome ICU = intensive care unit IF = immunofluorescent cytochemistry

IL-1RA = IL-1 receptor antagonist

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IP10 = interferon gamma induced protein 10 / CXCL10

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IL = interleukin

MCP = Monocyte chemotactic protein / CCL chemokine

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M-CSF = Macrophage colony-stimulating factor

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MIF = Macrophage inhibitory factor

MIG = Monocyte induced by gamma-interferon / CXCL9 MIP = Macrophage inflammatory protein / CCL chemokine

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NE = norepinephrine

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PDGF-bb = Platelet derived growth factor bb

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RANTES = Regulated and normal T cell expressed and secreted / CCL5 S-CGF-beta = Stem cell growth factor beta SCUF = slow continuous ultrafiltration SDF-1a = Stroma cell derived factor 1a / CXCL12 TER = transendothelial electrical resistance TNF = Tumor necrosis factor TRAIL = TNF related apoptosis inducing ligand VCAM-1 = Vascular cell adhesion molecule-1 VEGF = Vascular endothelial growth factor vWF = von Willebrandt factor

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Journal Pre-proof D EC LA R ATIO NS Ethics approval and consent to participate Ethical approval was waived due to the nature of a singular case report with data anonymized before publication. Consent for publication: Not applicable Availability of data and and materials:

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The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Conflict of interests:

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KS and SD received travel grants from CtoSorbents Corp. Otherwise, t he authors

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declare that they have no competing interests.

Not applicable

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Authors' contributions:

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Funding:

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SD und KS collected clinical data from the patient data monitoring system and generated the figures for publication. BMWS was the leading nephrology consultant coordinating and performing the cytokine adsorption. CK, GB, ME and AG were the leading hemato-oncologists performing the initial CAR-T-cell therapy and consulting physicians at the ICU. TS and NM were the leading neurology consultants assessing severity of CRES. SD and CSF performed the in vitro experiments and FACS analysis. SD, KS, CK, MMH and TW interpreted data, wrote the manuscript and calculated statics. SD had the original idea for this study. All authors read and approved the final manuscript. Acknowledgements:

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Journal Pre-proof Not applicable

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FIGU R ES

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21. Hawchar F, Laszlo I, Oveges N, Trasy D, Ondrik Z, Molnar Z. Extracorporeal cytokine adsorption in septic shock: A proof of concept randomized, controlled pilot study. J Crit Care. 2019;49:172-8. 22. Nishimoto N, Terao K, Mima T, Nakahara H, Takagi N, Kakehi T. Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease. Blood. 2008;112(10):3959-64. 23. Spooren A, Kolmus K, Laureys G, Clinckers R, De Keyser J, Haegeman G, et al. Interleukin 6, a mental cytokine. Brain Res Rev. 2011;67(1-2):157-83. 24. Nellan A, McCully CML, Cruz Garcia R, Jayaprakash N, Widemann BC, Lee DW, et al. Improved CNS exposure to tocilizumab after cerebrospinal fluid compared to intravenous administration in rhesus macaques. Blood. 2018;132(6):662-6. 25. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119(12):2709-20. 26. Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. 2014;6(224):224ra25. 27. Giavridis T, van der Stegen SJC, Eyquem J, Hamieh M, Piersigilli A, Sadelain M. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med. 2018;24(6):731-8. 28. Bonavia A, Groff A, Karamchandani K, Singbartl K. Clinical Utility of Extracorporeal Cytokine Hemoadsorption Therapy: A Literature Review. Blood Purif. 2018;46(4):337-49. 29. Peng ZY, Carter MJ, Kellum JA. Effects of hemoadsorption on cytokine removal and short term survival in septic rats. Critical care medicine. 2008;36(5):1573-7. 30. Kellum JA, Song M, Venkataraman R. Hemoadsorption removes tumor necrosis factor, interleukin-6, and interleukin-10, reduces nuclear factor-kappaB DNA binding, and improves shortterm survival in lethal endotoxemia. Critical care medicine. 2004;32(3):801-5. 31. Gruda MC, Ruggeberg KG, O'Sullivan P, Guliashvili T, Scheirer AR, Golobish TD, et al. Broad adsorption of sepsis -related PAMP and DAMP molecules, mycotoxins, and cytokines from whole blood using CytoSorb(R) sorbent porous polymer beads. PloS one. 2018;13(1):e0191676.

FIGURE 1 – Clinical course of CRS. Demonstrated are clinical parameters such as vasopressor and inotropic support, fluid balances as well as specific therapeutic measures (renal replacement therapy, tocilizumab and methylprednison infusion, CytoSorb adsorption) beginning at admission to the intensive care unit. Time points at the x-axis are marked in hours beginning from admission to the ICU. CRS - cytokine release syndrome, Dobu - Dobutamine, Epi - Epinephrine, Fluid marks daily total fluid balances and I-IV describe time points of additional blood sampling.

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FIGURE 2 – Ex vivo effect of the patient’s plas ma on endothelial morphology and function. (A) Ex vivo incubation of human umbilical vein endothelial cells (HUVECs) for 30 minutes with patients’ plasma obtained at time points 0, 8, 16, and 24hrs after start of extracorporeal cytokine adsorption.

Immunofluorescent

cytochemistry for the cell-cell contact protein VE-cadherin (green) and the cytoskeletal component f-actin (red) showing severe and progressive alterations of the endothelial architecture and the formation of paracellular gaps (i.e. the cellular correlate of the clinical capillary leakage syndrome; white arrows) (B) Transendothelial electrical resistance (TER) – a highly quantitative method to assess permeability in real-time in vitro – showing that patients’ plasma at distinct time

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Journal Pre-proof points after the start of cytokine adsorption differentially induce a drop in transendothelial resistance (TER). This drop in resistance was most noticeable at later time points (16 and 24hrs). 8h

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Journal Pre-proof TAB L ES TABLE 1 – Cytokines, chemokines and markers of endothelial activation that decreased during Cytosorb treatment. Plasma was obtained at time points 0, 8, 16, and 24hrs after start of additional treatment with cytokine adsorption. A significant decrease in concentration was defined as a reduction by at least 15% from baseline following 24hrs of cytokine adsorption. All concentrations are expressed in pg/ml.

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Table 1: Cytokines, chemokines and endothelial markers decreased during Cytosorb treatment 0h 8h 16h 24h % Δ 0% Δ max24h 24h Cytokines 44,85 45,64 24,96 9,38 -79,1 -79,4 IL-1 2,32 2,98 2,88 0,87 -62,5 -70,8 IL-1 4,17 2,43 4,56 0,61 -85,4 -86,6 IL-2* 1,14 1,56 1,86 0,51 -55,3 -72,6 IL-4* 41,56 85,07 53,36 13,97 -66,4 -83,6 IL-5 1345,55 1470,2 690,16 410,34 -69,5 -72,1 IL-6 12,35 13,02 8,18 6,01 -51,3 -53,8 IL-7 52 64,68 50,08 26,08 -49,8 -59,7 IL-9 11,37 14,91 13,97 7,36 -35,3 -50,6 IL-10 4,23 3,00 2,50 OOR < IL-12p70* 599,65 629,22 1348,84 412,36 -31,2 -69,4 IL-12p40 6,79 6,36 4,71 1,18 -82,6 -82,6 IL-13 68,24 74,43 47,27 *3,18 -95,3 -95,7 IL-15 531,24 545,88 267,41 155,99 -70,6 -71,4 IL-16 9,16 8,43 8,18 3,19 -65,2 -65,2 IL-17* 30,53 33,25 21,06 10,63 -65,2 -68,0 TNF- 25,76 32,79 29,41 12,79 -50,3 -61,0 TNF- 124,15 177,53 132,21 73,85 -40,5 -58,4 Interferon- 6,91 6,91 7,77 3,56 -48,5 -54,2 Interferon-2* Chemokines 16,5 15,35 18,96 8,02 -51,4 -57,7 Eotaxin/ CCL11 25,98 25,51 28,71 15,09 -41,9 -47,4 FGF-b 44,84 42,93 39,13 36,2 -19,3 -19,3 CCL2/MCP-1 3,62 5,24 4,98 *0,53 -85,4 -89,9 CCL7/MCP-3 7,74 6,86 8,58 6,37 -17,7 -25,8 CCL3/MIP-1 59,98 57,69 63,86 36,01 -40,0 -43,6 CCL4/MIP-1 454,5 499,82 587,7 202,43 -55,5 -65,6 CCL5/RANTES 605,18 625,66 438,06 204,94 -66,1 -67,2 CCL27/CTACK 550,24 530,67 462,09 266,99 -51,5 -51,5 CXCL1/GRO-a 8511,1 9060,65 11038,93 5829,48 -31,5 -47,2 CXCL9/MIG

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Journal Pre-proof 13617,53 217,7 234,37 124,72 145,28 36,75 2072,7

10741,44 208,71 185,15 177,57 149,03 47,37 2086

19054,68 129,13 259,05 142,86 120,28 48,74 1509,02

10745,06 61,89 158,79 96,73 45,64 27,44 902,81

-21,1 -71,6 -32,2 -22,4 -68,6 -25,3 -56,4

-43,6 -71,6 -38,7 -45,5 -69,4 -43,7 -56,7

148,48 6148,45 3949,13 42894,64

158,76 6502,53 3552,36 48669,01

80,99 690,74 3357,95 69757,86

19,96 888,99 2960,85 31854,67

-86,6 -85,5 -25,0 -25,7

-87,4 -86,3 -25,0 -54,3

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CXCL10/IP10 CXCL12/SDF-1a G-CSF M-CSF SCF TRAIL CD25/IL-2R Markers of endothelial activation VEGF Angpt-1 Angpt-2 ICAM-1

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ABBREVIATIONS: IL – Interleukin, TNF – Tumor necrosis factor, FGF – Fibroblast growth factor, MCP – Monocyte chemotactic protein, CCL – Chemokine ligand, CXCL – C-X-C motif chemokine, CTACK – Cutaneous T-cell attracting chemokine, MIG – Monocyte induced by gamma-interferon, IP10 – interferon gamma induced protein 10, MIP – Macrophage inflammatory protein, RANTES – Regulated and normal T cell expressed and secreted, SDF-1a – Stroma cell derived factor 1a, M-CSF – Macrophage colony-stimulating factor, Hu-SCF – Human stem cell factor, TRAIL – TNF related apoptosis inducing ligand, VEGF – Vascular endothelial growth factor, Angpt – Angiopoietin, ICAM-1 – Intercellular adhesion molecule-1, OOR< - Out of Range Below, * concentrations were close to the detection limit of 1-5 pg/ml

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TABLE 2 – Cytokines, chemokines and markers of endothelial activation that remained unchanged or increased during Cytosorb treatment. Plasma was obtained at time points 0, 8, 16, and 24hrs after start of additional treatment with cytokine adsorption. A significant decrease in concentration was defined as a reduction by at least 15% from baseline following 24hrs of cytokine adsorption. All concentrations are expressed in pg/ml.

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11,97 2424,41 165604,91

37,63 3072,23 369813,07

24,86 2195,85 270776,9

71,3 -4,4 55,6

148,56 688,72

247,96 881,88

240,82 13366,39

206,6 1838,56

39,1 167,0

0,64

0,55

4,86

3,33

420,3

*61138,00

*64190,72

*137377,80

124,7

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14,51 2296,84 174019,06

*241582,72

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Chemokines and ligands CXCL8/IL-8 MIF S-CGF- PDFG-bb HGF Markers of endothelial activation Angpt2/Angpt-1 VCAM-1

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Table 2: Cytokines, chemokines and endothelial markers that remained unchanged or increased during Cytosorb treatment 0h 8h 16h 24h % Δ 0-24h Cytokines 231,17 236,61 492,08 476,22 106,0 IL-1RA 149,08 255,02 735,59 676,49 353,8 IL-18

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ABBREVIATIONS: IL – Interleukin, IL-1RA – IL-1 receptor antagonist, CXCL – C-X-C motif chemokine, MIF – Macrophage inhibitory factor, G-CSF –Granulocyte colonystimulating factor, S-CGF-beta – Stem cell growth factor beta, PDGF-bb – Platelet derived growth factor bb, HGF – hepatocyte growth factor, Angpt – Angiopoietin, VCAM-1 – Vascular cell adhesion molecule-1, * - Value extrapolated beyond standard range

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