key: cord-0273093-2qw00hlv authors: Hanaoka, Shingo; Saijou, Shinji; Matsumura, Yasuhiro title: A novel and potent thrombolytic fusion protein consisting of anti-insoluble fibrin antibody and mutated urokinase date: 2020-09-08 journal: bioRxiv DOI: 10.1101/2020.09.06.284596 sha: 67cf297a01b20854c082c81978c97a7cad0d419e doc_id: 273093 cord_uid: 2qw00hlv Because the risk of thromboembolism increases with age, as well as due to infectious diseases, safer and more effective thrombolytic agents are in greater demand. Tissue plasminogen activator (tPA) is currently used clinically because it has higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity in places other than IF. Meanwhile, UK has the advantage that it is specifically activated on IF, but it only binds IF weakly. Unlike the anti-IF monoclonal antibody (mAb) established in the past, our anti-IF mAb recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the systemic side effects of UK in vivo. In a mouse thrombus formation experiment, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA, and 90% (9/10) in the AMU1114 treatment group. These data support future clinical development of AMU1114. Hypercoagulation occurs not only in cardiovascular diseases, but also in cancer and severe infectious diseases such as influenza and coronavirus infection, worsening their pathologies. [1] [2] [3] In patients with such severe conditions, administration of thrombolytic agents should be carried out with caution, and safer forms of administration are desirable. Currently, tissue plasminogen activator (tPA) is the thrombolytic agent used most commonly in clinics around the world because it binds IF more specifically than UK. 4 However, even under tPA treatment, bleeding is a clinically serious side effect. 5 To address this issue, efforts have been made to increase the fibrinolytic activity of plasminogen activators by selectively targeting them to IF in lesions. For these purposes, a mAb against IF called 59D8 is utilized as a delivery tool. 6 For example, some groups prepared a chemical conjugate of pro-UK with this mAb. 7, 8 Another group produced a recombinant fusion protein of the catalytic domain of UK and the scFv of 59D8. 9 Still other groups developed chemical conjugates of tPA and 59D8. 10, 11 However, for several reasons including low yield, inconsistent coupling, and low superiority relative to the original plasminogen activators, none of the fusions or conjugates were evaluated clinically. In addition to technical problems related to chemical conjugation and protein fusion, the 59D8 mAb used in those groups bound not only IF but also fibrinogen, which circulates in the blood. Therefore, it is assumed that those formulations were not efficiently delivered to the 4 lesion because they were neutralized by the large amounts of fibrinogen in the blood. As part of our research into cancer and blood coagulation, we established a mAb (102-10) that recognizes only IF and not fibrinogen, soluble fibrin (fibrin monomer), or soluble fibrin degradation products (FDP). 12, 13 The epitope of 102-10 is on the β chain that underlies the pit structure formed by the hydrophobic regions of the β and γ chains, which is exposed only when IF is formed. Because fibrinogen, the fibrin monomer, and FDPs are soluble, the pit structure is completely closed in aqueous solution due to hydrophobic bonding; consequently, 102-10 does not bind soluble molecules such as fibrinogen, the fibrin monomer, or FDP. The amino acid sequence of this epitope is widely conserved among animals ranging from fish to humans. In other words, even though 102-10 is an anti-human IF antibody produced in mice, it also recognizes mice IF. This suggests that data from mouse experiments can be extrapolated to humans. Even single-chain tPA (pro-tPA) has enzymatic activity that converts plasminogen in circulating blood into plasmin. 14, 15 Plasmin and activated tPA in the blood are inhibited by innate α 2-plasmin inhibitor (α2-PI) 16 and plasminogen activator inhibitor-1 (PAI-1), 17, 18 respectively. On the other hand, pro-UK is rarely activated naturally in blood circulation and is not inhibited by PAI-1. 19 Consequently, UK is active only on IF in the lesion, where plasmin is abundant. Based on these observations, we hypothesized that a thrombolytic agent superior to tPA could be obtained if it were possible to efficiently deliver pro-UK to IF. Thus, we have prepared a fusion protein of pro-UK and anti-IF mAb to deliver pro-UK selectively to IF in lesions in the body. The fibrinogen β -chain D domain (a.a. 228-491, UniprotKB entry number P02675) was expressed in E. coli and used as an immunogen. The antigen, mixed with adjuvant, was administered four times intraperitoneally to BALB/c mice, followed by final immunization through the tail vein. Three days after the final immunization, the spleen was removed, and the spleen cells were fused with X63 myeloma cells by the PEG method to obtain an antibody-producing hybridoma. Hybridomas that were immunogen-positive, IF-positive, and fibrinogen-negative were screened by ELISA. The clone producing an antibody with the highest binding strength and specificity was established and named 1101. The isotype of the antibody was determined using an isotype-specific anti-mouse antibody measurement kit (Bethyl). An antibody that was unreactive to both IF and fibrinogen was used as a negative control. Cloning was performed by 5'-RACE, and the gene sequence was confirmed. The confirmed CDR region was inserted into human IgG (Human Monoclonal Antibodies-Methods and Protocols, Springer Protocols). Humanized 1101 and control antibodies were expressed in CHO cells, and IF specificity was confirmed by ELISA. Preparation of IF-immobilized plates. Fibrinogen (Sigma-Aldrich) was immobilized on 96-well plates at a concentration of 1 μ g/well and allowed to stand overnight at 4°C. One hundred microliters of 0.05 U/mL thrombin (Sigma-Aldrich), 7 mM L-cysteine, and 1 mM CaCl 2 was added to each well, and the plates were incubated at 37°C for 2 hours. After blocking with N102 (Nichiyu) containing 10% sucrose, blocking was performed again with TBS-T (Tris-buffered saline with Tween-20) containing 1% BSA, yielding IF-immobilized plates. Blood was collected in a vacuum blood collection tube (Venoject II VP-P070K30, Terumo) and Statistical analyses of ELISA assay, fibrinolytic assay, and plasma level of plasminogen were performed using the EZR software. P-values were determined by analysis of variance (ANOVA) and Tukey's test. In animal experiments, we compared the control with tPA and control with AMU1114. We also compared tPA and AMU1114 in terms of the number of cases in which blood vessels were patent at the end of the experiment. The time to vascular occlusion and the patency rate of blood vessels were tested by the F test (significance level: 5%) for uniformity of variance. For the number of cases in which blood vessels were patent at the end of the experiment, a χ 2 test was performed. If the variances were uniform, Student's t test was used for comparison. If the variances were not uniform, the comparison was performed using the Aspin-Welch t-test. Significance levels were set to 5% and 1%, and a two-sided test was performed. For the evaluation data, the average value ± standard error of each group was obtained. In addition, individuals whose blood vessels were patent at the end of the experiment were counted. The hand, mAb 59D8, which recognizes the N-terminus of fibrinogen cleaved by thrombin, recognized not only IF but also fibrinogen 6 ( Figure 1B) . Like 102-10, 1101 bound not only human IF but also mouse IF ( Figure 1C ). In addition, the binding strength and specificity of 1101 for IF was higher than that of 102-10 (data not shown). Immunohistochemistry with 1101 revealed clear IF deposition in human thrombosis ( Figure 1D ). In the IF formation assay, anti-thrombin III (AT III) suppressed IF formation. On the other hand, 1101 caused IF formation to the same extent as control PBS. In the fibrinolysis assay system, α 2-plasmin inhibitor (α2-PI) and plasminogen activator inhibitor 1 (PAI-1) clearly decreased fibrinolytic activity. As in the control, 1101 did not delay fibrinolysis. These results indicated that the coagulation and fibrinolytic systems were unaffected by 1101, as with the previously established antibody 102-10 ( Figure 1E ). 21 1 6 A portion of UK was linked to the Fab region of the H chain of humanized antibody 1101 via a 16-residue linker. An antibody-UK fusion protein was generated for two types of UK (Figure 2A) . Figure 2B ). The mutated UK did not decrease fibrinolytic activity; the in vitro fibrinolytic activity of AMU1114 was similar to that of UK and construct 1 ( Figure 2C ). When comparable molar quantities of UK, tPA, and AMU1114, were applied to fibrin gels, UK, tPA, and AMU1114 all showed significantly stronger fibrinolytic activity than the control (P < .001), and fibrinolytic activities were similar among the three agents (P > .5) ( Figure 3A ). Plasma plasminogen levels after administration of PBS, UK, tPA, and AMU1114 were 94.7%, 51.1%, 74.9%, and 85.5%, respectively, relative to the level in plasma of untreated mice. UK administration significantly decreased plasma plasminogen relative to the other groups (UK vs tPA, P < .05 and UK vs AMU1114, P < .01). There was no significant difference in plasminogen levels between tPA and AMU1114 treatment. However, plasma plasminogen was significantly lower in the tPA group than in the untreated group (P < .01), and there was no significant difference between the AMU1114 and untreated groups ( Figure 3B ). In the PIT experiment in the mouse carotid artery, 22 the times from the start of green light irradiation to complete obstruction of blood flow in the control, tPA, and AMU1114 groups were 553.1 ± 141.9, 1135.7 ± 308.3, and 1267.8 ± 308.2 seconds, respectively. Multiple comparisons were performed among the three groups, but no significant differences were detected ( Figure 4A ). Rates of vascular patency for 60 minutes were 21.1 ± 3.7%, 50.9 ± 10.1%, and 66.3 ± 7.2% respectively, in the control, tPA, and AMU1114 groups. The rate of vascular patency for 60 minutes in the tPA and AMU1114 treatment groups was significantly higher than that in the control ( Figure 4B ). In addition, when multiple comparisons were performed among the control, tPA, and AMU1114 groups, significant differences were detected between the control and tPA group (P < .05) and between the control and AMU1114 group (P < .01), but there was no significant difference between the tPA and AMU1114 groups, although AMU1114 tended to have a higher rate of vascular patency than tPA. At the end of the measurement, the number of mice with open blood vessels was 0/10 in the control group, 5/10 in the tPA group, and 9/10 in the AMU1114 group ( Figure 5A ,B). Significant differences in the number of individuals whose blood vessels were patent were detected between the control and tPA groups (P = .0098), and between the control and AMU1114 groups (P = .0001). On the other hand, no significant difference was detected between the tPA and AMU1114 groups, although AMU1114 tended to have a stronger thrombolytic effect than tPA (P = .051). 1 9 In this study, we successfully constructed a fusion protein of mutated UK and anti-IF mAb, AMU1114, and confirmed that it retained the activity of UK. When UK and AMU1114 were administered to mice, the reduction in plasminogen was more strongly suppressed by AMU1114 than by UK, indicating that AMU1114 is safer than UK. The in vitro fibrinolytic activity of AMU1114 was almost the same as that of tPA. The bleeding toxicity was lower for AMU1114 than for tPA, according to the in vivo experiments of the effect of test substances to plasminogen consumption in blood of normal mice. In terms of the antithrombotic effect, a thrombus was formed in the carotid artery of mice by the PIT method, and the thrombolytic effect of the test substances was examined. Among the control, tPA, and AMU1114 groups, there was no significant difference in the time from the start of green light irradiation until complete occlusion of blood flow (TTO), although tPA and AMU1114 took longer than the control. Regarding vascular patency, tPA and AMU1114 had longer vascular patency times during blood flow measurements than the control. At the end of blood flow measurement, the number of individuals whose blood vessels were patent was 0/10 for controls, 5/10 for tPA, and 9/10 for AMU1114. Thus, tPA and AMU1114 had the effect of thawing the prepared thrombus and reopening blood vessels. At the same time, AMU1114 had a greater thrombolytic effect than tPA, as reflected by the larger number of individuals with reopened blood vessels. Our They may also be associated with worsening of clinical condition, e.g., fibrosis associated with elevated blood coagulation at local sites of infection and cancer. Although it may be difficult to use a 1 thrombolytic agent in such situations, we believe that developing a safer formulation is a step in the right direction. While additional data are accumulated, a research cell bank of this antibody-UK fusion protein should be established for the purpose of future clinical trials in patients with potentially fatal coagulopathy associated with cerebral infarction, myocardial infarction, cancers, and viral infection. Y.M. is co-founder, shareholder, and Board Member of RIN Institute, Inc., a venture company spun 8 Effects of various thrombolytic agents on plasma plasminogen levels in mice (n = 3). The amount of plasminogen in each plasma sample was measured, and the ratio relative to the amount of plasminogen in untreated mice is shown. Statistical analysis was performed using Tukey's test. *P < .05. **P < .01. ***P < .001. 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