TW520289B - Anti-selectin antibodies for prevention of multiple organ failure after polytrauma and for prevention of acute organ damage after extracorporeal blood circulation - Google Patents

Abstract

Anti-selectin antibodies are used for reducing probability of incidence of polytraumatic events, such as organ failure. A pharmaceutical composition for treating polytraumatic event, comprising a therapeutically effective amount of an anti-selectin antibody in combination with a pharmaceutically acceptable carrier.

Description

520289 A7 B7 5. Description of the invention (i) The present invention relates to the use of anti-selectin antibodies to prevent most organ failures related to multiple trauma, and to prevent acute organ failure related to extracorporeal blood circulation. Particularly preferred are antibodies to E-selectin, L-selectin, and / or P-selectin. BACKGROUND OF THE INVENTION Multiple trauma is known as damage to many tissues (bone or soft tissue). During multiple trauma events, mediator systems (such as cytokines, arachidonic acid products, oxygen free radicals, proteases), as well as white blood cells and other cells are activated. This can cause secondary organ damage (such as tissue damage caused by released proteases). This secondary organ injury can occur throughout the body, regardless of the location of the primary injury. Multiple trauma may also be associated with hemorrhagic shock. Hemorrhagic shock is finally understood as a shock characterized by rapid and considerable loss of blood inward or outward. Currently, hemorrhagic shock can be successfully treated with medical treatment, especially fluid replacement and blood transfusion. Hemorrhagic shock plus trauma is called hemorrhagic traumatic shock. In contrast to pure hemorrhagic shock, there is currently no specific treatment for traumatic or hemorrhagic traumatic shock, and there is no way to prevent organ failure after multiple traumas. Most organ failure (MOF) is a serious problem that often occurs in multiple trauma. The more organs damaged, the higher the mortality rate. Organs and systems that fail include the heart, lungs, kidneys, liver, stomach, intestine, and central nervous system. Although in recent years through the improvement of rescue services and emergency medical care, the extremely high mortality rate of trauma patients has been reduced by about 20%, but the paper size of the organ is applicable. National Standard (CNS) A4 specification (210X297 mm) 4 — (Please read the notes on the back before filling this page) Order printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 520289 A7 ____B7__ 5. Description of the invention (2) There is no specific treatment for exhaustion. Ma r z i et al., J. T r a u m a 35: 110-119 (193 9) revealed that superoxide dismutase can be administered 24 hours after trauma. However, the results are not clear, and there is only a slight tendency for partial improvement. However, no substantial reduction in mortality has been observed. Mileski, W. J. et al., Surgery 108: 206 — 212 (1990) revealed that the combination of neutrophils or their aggregates can substantially cause the development of hemorrhagic shock following organ damage. In this example, the experimental animals were given anti-CD18 antibody immediately after the 90-minute hemorrhagic shock period. Mileski does not describe treatments for most organ failures after multiple trauma. Vedder N.B. et al., Surgery 106: 509-516 (1989) also proposed the use of anti-CD 18 antibody for the treatment of hemorrhagic shock. Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page). It was found that selectins, such as L, E, and P-selectin, are related to tissue damage during hemostasis and reperfusion. The neutrophil plays an important role in this connection. It is assumed that selectin is necessary for neutrophil supplementation. Obviously L-selectin is necessary for the complete development of osteoclastic muscle and lung injury. (Seekamp a. Et al., Am. J. Pathol. 1 1: 5 9 2 -598 (1994). Mulligan, MS et al., J. Immunol. 151: 832 — 840 (1994) describe a similar phenomenon e The production of humanized anti-L-selectin antibody is described in WO 94/1 2 2 1 5 and has been listed as a reference. It is proposed to use this antibody to treat inflammatory diseases, especially myocardial infarction. Build 1-5 mg The dose can prevent acute lung failure. However, the reference dose is not described in the prevention of multiple trauma. The paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) ~ _ -5-520289 A7 _B7___ 5. Description of the invention ( 3) The method of MOF. (Please read the precautions on the back before filling out this page) Therefore there is a need for effective therapies to prevent and / or treat most organ failures after multiple trauma. Acute organ injury can also be taken into consideration Caused by vascular surgery, such as aorta-coronary venous circuit surgery or heart valve surgery, where the patient's blood is circulated extracorporeally through a cardiopulmonary machine. The degree of injury depends on the operation of the machine. This can cause lung failure, which can cause after surgery Respiration is necessary (B i rnbaura, D. eta 1., Z. Kardiol. 79, Suppl. 4: 87-93 (1990)). Other organs such as the heart, kidney, liver, or systems such as blood and blood coagulation The system can also be damaged and fail. Known by Mulligan, MS eta 1., J. Immunol. 1 5 1: 832 — 840 (1994), molecules that promote adhesion, such as L, E, and P-selectin It is involved in the process of acute inflammation. These molecules can mediate the adhesion interaction between leukocytes and endothelial cells. In this relationship, L-selectin appears to play an important role in the initial phase (rolling phase) of the acute lung luminescence response. Mulligan further stated that anti-L-selectin antibodies are suitable for shortening the period of lung injury initiated by L-selectin. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economics, and so far, no preventive therapies are known and can be used In order to prevent acute organ injury caused by extracorporeal blood circulation, effective therapy is needed to prevent acute organ injury caused by extracorporeal blood circulation. OBJECT OF THE INVENTION The object of the present invention is to propose a method and a treatment group. It can be used to effectively prevent most organ failure after multiple trauma in the human body, and it can be used to a considerable extent. This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) ~ -6-520289 A7 B7 V. Description of the invention (4 ) To reduce mortality in patients with multiple trauma. The present invention relates to the therapeutic use of anti-selectin antibodies and the production of pharmaceutical compositions that can be used to prevent multiple organ failure and multiple trauma deaths. The object of the present invention is also to propose a method containing an anti-selectin antibody and the use of a therapeutic composition to prevent acute organ injury after extracorporeal circulation. Such organ damage can be largely avoided by this method and procedure. A special advantage of the method is the in vitro application, which can cause organs and disease to effectively reduce β. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the wet weight of the lungs of experimental animals at the observation time. Figure 2 shows the cardiovascular variables C0 (cardiac output) of various experimental animals over time. Figure 3 shows the cardiovascular variable MAP (mean arterial blood pressure) of experimental animals over time. Figure 4 shows the B E value of the experimental animals over time (the arteries exceed the baseline value). Figure 5 shows the number of white blood cell cells in various experimental animals over time. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling out this page) Detailed description The present invention is related to the use of at least one anti-selectin antibody to produce pharmaceutical compositions to prevent patient blood from passing through the heart and lung machines Acute organ injury after extracorporeal circulation, in which anti-selectin antibodies are added to the heart and lung machine's pipeline system in vitro from 1 to 30 minutes before the end of extracorporeal circulation. The dosage is 1.0-10 mg per kilogram of patient's body weight, And preferably 2-4 milligrams. This paper scale is applicable to China National Standards (CNS) A4 specifications (210X mm). Ί 520289 A7 B7 5. Invention description (5) / kg. Surprisingly, with this preventive in vitro administration, acute organ injury after extracorporeal circulation of the patient's blood can be largely prevented. In a preferred embodiment, a total of 1-4 mg / kg anti-selectin antibody, such as anti-L-selectin antibody, is administered to the patient in a further dose within 1 to 3 days. Multiple or single strains, murine, human, chimeric or humanized antibodies / immunoglobulins and their binding fragments can be used as anti-selectin antibodies. In one aspect of the invention, the therapeutic composition is not administered to a patient, but is administered externally, that is, directly into the piping system of a cardiopulmonary machine. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). ≫ Anti-selectin antibody 'used in this context means any antibody that can bind to selectin. Particularly preferred are antibodies that specifically bind to one of L-selectin, E-selectin or P-selectin, and combinations thereof. Antibodies that can react with more than one selectin, such as antibodies that can react with both L- and E-selectin, are also preferred. L-selectin is a known glycoprotein that can be constitutively expressed for all white blood cells. L-selectin and its murine homologues, GP 90 and Me 1 1 4, are involved in the normal recirculation of lymphocytes' and they each raise circulating lymphocytes and blood vessels in high endothelial veins (HEVs) of lymphoid organs Interactions between ligands (also called requestors ~ addressins ") (LA Lasky, et al ·, Cell 6 9: 9 2 7- 9 38 (1992); Ε · L. Berg, et al ·, J Cell Biol. 114: 343-349 (1991)). In addition to its role as a lymphocyte regression receptor, L-selectin is also involved in the adhesion of white blood cells to non-lymphoid tissues in circulation, such as the endothelium in luminescence. L-selectin can be detached from the surface of white blood cells after activation of white blood cells (T · K · Kishimoto, et al., Science 245: 1238- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm)-8- 520289 A7 _B7_ 5. Description of the invention (6) 124 1 (1 98 9)), and this is an important process in retaining activated white blood cells on the light-emitting site. L-selectin has an amine-terminated carbohydrate confirmation region (CRD) that is quite homogeneous to C-type exogenous lectin (K. Trickhamer, J. Biol. Chem. 2 6 3 9 5 5 7-9 5 6 0 (198 8)), followed by a single epithelial growth factor region, a complement regulatory region, a single transmembrane polypeptide and a carboxy-terminal cytosolic region. L-selectin interacts with its cognate ligand via an amino-terminal CRD in a calcium-related manner. According to the present invention, the anti-selectin antibody is better controllable and can better inhibit the interaction between the CRD region and the equivalent carbohydrate receptor on the cell surface. Such a carbohydrate receptor is described by R. B. Parekh, Tibtech 12: 339-345 (1994) and is listed as a reference. These carbohydrate receptors can be phosphorylated or sulfated sugars. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) In a further specific example of the present invention, anti-P — and / or anti-E —selectin antibodies can be used instead , Or add anti-L-selectin antibodies. Such antibodies can be produced using P- or E-selectin (described in R. B. Parekh and T. F. Tedder, FASEB Journal 9: 866-873 (1995), which is incorporated herein by reference). In a particularly preferred embodiment of the present invention, anti-P- and / or anti-E-selectin antibodies are used, which can show comparable cross-reactivity with L-selectin antibodies, especially with antibodies HuDreg-55 or HuDreg Cross-reactivity of -200. As used herein, a humanized immunoglobulin # refers to an immunoglobulin containing a human structure, at least one from the complementarity determining region of a non-human antibody. The paper size is applicable to the Chinese National Standard (CNS) 8-4 (210X29? Mm). ) -9-520289 A7 B7 V. Description of the invention (7) (CDR), and any existing fixed region is substantially the same as the human immunoglobulin fixed region, that is, at least about 8 5-9 0%, preferably at least 9 5% are the same. Therefore, all parts of the humanized immunoglobulin, except for possible CDRs, are substantially identical to a considerable portion of more than one natural human immunoglobulin sequence. For example, humanized immunoglobulins will not include embedded mouse variable region / human fixed region antibodies. See, for example, European Patent EP A 451216, which has been incorporated by reference in this case. The invention also relates to the use of such anti-selectin antibodies to reduce MOF and mortality after multiple trauma. Surprisingly, it is possible to avoid most organ failures when anti-selectin antibody ', especially anti-L-selectin antibody, is administered very quickly after multiple trauma. It is also very surprising that no actual symptoms develop at this early stage, and therefore there is no reason to administer this dose as a precautionary measure. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). It is also very surprising to know that the dose of anti-selectin antibody is 1.0--0 0 mg / kg, preferably 2- 4 mg / kg, it is beneficial to administer 1 to 5 times, preferably 1 or 2 times after multiple trauma. The first application is as early as possible, preferably 0.5 to 8 hours after the multiple trauma event. , Particularly preferably 0 · 5-4 hours. Individual application intervals are between about 6 and about 72 hours, preferably between 6 and 36 hours. In a preferred embodiment, the secondary and subsequent doses and time may depend on the concentration of anti-selectin antibody in the blood, preferably the concentration in plasma or serum, which is a variable that can be determined at an early stage. In this connection, the plasma concentration of the better anti-selectin antibody is maintained at 10-100 micrograms / ml, which lasts 7-10 days after the multiple trauma event. This concentration is equivalent to the Chinese paper standard (CNS) Λ4 specification (210 × 297 mm) in blood plasma. 10-520289 A7 __ ^ _ B7 V. Description of the invention (8) Soluble selectin concentration 1 0-1 0 0 Times excess. The dose and time of secondary and subsequent administration can be determined at 6-24 intervals by determining the concentration of anti-selectin antibodies in blood, serum or plasma, and administered immediately when the plasma concentration drops to 10 μg / ml antibody Substantially equivalent to the first dose. When the antibody concentration is between 10 and 50 μg / ml, about half of the antibody at the first administration is administered, and no further antibody is administered at the antibody concentration of 50 and 100 μg / ml . In this case only the antibody concentration was tracked further. The concentration of anti-selectin antibodies in blood, serum, or plasma is determined by a general method, preferably an immunological determination method. This method is known to the artisan. The determination can be made using the ELISA test, in which the labeled selectin-specific antibody, preferably the antibody that is also used therapeutically, competes for the specific selectin. In the next step, the labeled antibody content bound to the antigen is determined, and the concentration of anti-selectin antibody in the sample is determined from this. The therapeutic composition of the present invention is usually administered parenterally, such as intravenously, intra-arterially, intraperitoneally, subcutaneously or intramuscularly. Intravenous (i.v. printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page)) as the active component in the preferred β composition can be used in liquid or solid form , Preferably in a freeze-dried form, and used with a suitable diluent or carrier, such as water or sodium chloride, dextrose, an aqueous solution of a buffer substance, and the like. Other suitable medicinal auxiliary substances can also be added. Antibodies to selectins can be known from technical statements and described, for example: ΕΡ—Α 0 3 8 6 9 0 6 * W 0 93/00111 and W 0 94/12215, and Kishimoto, T.K. et ai. , Blood 78: 8 0 5- 8 1 1 (1 9 9 1) and Proc · Natl · Acad · Sci. This paper size is applicable to China National Standard (CNS) A4 specification (210X 297 mm) " 1-11 1 1 520289 A7 B7 V. Description of the Invention (9) USA 87: 2241-2248 (1990), all of which are listed for reference. In the literature, L-selectin is also called LECAM-1, Me1 14 or Lam (please read the notes on the back before filling this page). The selection and sequence of Lam-1 has been described in WO 9 3/0 2 6 9 8. An antibody capable of specifically binding to a selectin is suitable. Humanized antibodies are suitable, especially HuDreg 200, which is described in WO 94/12215 and listed for reference herein. Other antibodies that can bind to selectin, such as HuDr eg 55 (sequences: SEQ ID NOs: 1 to 4) are also particularly preferred. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. The a-antibody used in this is known as a protein, which is composed of more than one polypeptide chain, which is basically encoded by the antibody gene. Antibody genes direct the synthesis of antigen-specific variable regions, and may also direct the synthesis of fixed regions of genes. It is understood in the sense of the present invention that antibodies can be various derivatives and fragments of antibodies, such as FV, Fab and F (ab) 2, and individual antibody chains (Houston et al., PNAS USA 85 5 8 79-5883 ( 1988), Bird et al., Science 242: 423-426 (1988), Hood et a 1., Immunology, Benjamin NY, 2nd edition (1984), Hunkapiller and Hood, Nature 323 15-16 (1986)). Monoclonal antibodies and fragments thereof, and particularly chimeric or humanized antibodies of the IgG1 or IgG4 subtype are preferred. The antibody preferably contains at least two light polypeptide chains and two heavy polypeptide chains. Each of these chains contains a variable region (usually the N-terminal portion of a polypeptide chain), which in turn contains regions that can bind to an antigen. The heavy and light chains may additionally contain a fixed region of the polypeptide (usually the C-terminal portion) that mediates the binding of antibodies to white blood cells (neutrophils, lymphocytes, etc.). Generally, the paper size of the light and heavy chains is in accordance with the Chinese National Standard (CNS) A4 (210X 297 mm)-12-520289 A7 B7 V. The description of the invention (ίο) is a complete antibody chain, which consists of a variable region and a complete Composed of fixed areas. In this connection, the variable and fixed regions can be derived from different antibodies, such as different isotypes. For example, a polypeptide containing the variable region of an anti-selectin antibody r-1 isotype heavy chain may be linked to a fixed region from the heavy chain of another type (or subtype) of antibody. Anti-selectin antibodies in which more than one amino acid is replaced are also suitable. In this example, the amino acid is preferably replaced with another amino acid having similar characteristics (for example, the acid amino acid Asp is replaced by the acid amino acid G 1 u). This substitution does not change the structural characteristics of the original sequence. Examples of such polypeptide structures are described in Proteins, Structures and Molecular Principles, Creighton (editor), WH Freeman and Company, New York (1894); Introduction to Protein Structure, C. Brandon and J. Tooze, Garland Pub 1 is shing, New York (1981); Thornton et al., Nature 354 105 (1991). In general, antibodies suitable as anti-selectin antibodies are those that can bind more than one of L-selectin, E-selectin, and P-selectin and / or can suppress fluctuations in white blood cells (such as neutrophils) . Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling out this page) In addition to the humanized immunoglobulins described here, it is easy to use various recombinant DNA technologies well known to the artisan To design and manufacture other ~ substantially homogeneous " modified immunoglobulins. Human antibodies ' including, for example, Eu or GAL antibodies, as well as other human antibodies known in the art, can be used as sources of architectural sequences. These structural sequences should be highly sequence homogeneous with the mouse Dr eg 55 or mouse Dr eg 200 variable regions from which CDRs are derived. The heavy and light chain variable architecture regions can be derived from the paper size applicable to China National Standard (CNS) A4 specifications (210X 297 mm)-13-520289 A7 ____B7 V. Description of the invention (11) The same or different human antibody sequences. Indeed, each of the heavy and light chain framework regions can be derived from more than one human antibody. The human antibody sequence may be a naturally occurring human antibody sequence, or it may be the consensus sequence of many human antibodies. See Carteretal, WO 92/22653 (1992), which has been listed for reference in this case. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling out this page) A. Antibodies that can be bound in a considerable way are known to be antibodies that can bind to the same or overlapping epitopes. Epitope overlap can be determined by methods known in the art, such as with the help of competitive testing systems. A competitive binding assay can be performed to determine this, and determine the extent to which the antibody will compete with HuDr eg 55 for binding to the solidified L-selectin antigen. For this, L-selectin (preferably L-selectin on white blood cells) solidified in a suitable manner was incubated with labeled HuDr eg 55, and then tested for excess antibody. Determine the degree of binding of the antibody to L-selectin to be tested and compare it with HuDr eg 55. This is to determine the combined flag in the anti-leukocyte-binding flag. If the labeled Hu D r e g 5 5 is at least 50% replaced by the antibody to be tested, this indicates the presence of overlapping epitopes. Antibodies that bind to HuD r g 5 5 in a comparable manner are preferred in the present invention. > Antibodies that can be combined in a comparable manner can also be identified by screening for the ability to block neutrophil-endothelial cell interaction. A simple visual analysis to detect such interactions is described by Kishimoto et al. (Blood, 78: 805 (1991)). In brief, a single layer of human umbilical vein cells was stimulated with melatonin-1. The neutrophil was pretreated in the test with or without antibodies, added to a single piece under clear conditions, and then examined with a microscope to determine the paper size. Applicable to China National Standard (CNS) A4 (210X 297 mm). -14-520289 A7 B7_ 5. Description of the Invention (l2) Determine the number of neutrophils to be attached. In one approach, neutrophils can be obtained from patients lacking human leukocyte adhesion. See Anderson et al., Ann. Rev. Med. 38: 1 7 5 (19 8 7). The neutrophil from this patient lacks the integrin receptor, and its binding to the neutrophil may obscure the effect of blocking L-selectin binding. The antibody may be a user having a complete monoclonal antibody, a fragment thereof (Fv, (Fv) 2, Fab /, F (ab >) 2), a chimera, a humanized or a human antibody. Short antibody fragments containing only a portion of the CDR region, but specifically binding to L-selectin can also be used. Antibodies, especially monoclonal antibodies and fragments thereof, are well-known by the artisan and are described in, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988), Bessler et al. al., Immunobiol. 170: 239-244 (1985), Jung et al.,, Angewandte Chemie '97: 883 (1985), Cianfiglia et al., Hybridoma Vol. 2: 451-457 (1993) Printed by the Consumer Bureau of Standards Bureau (please read the notes on the back before filling out this page) The anti-selectin antibodies that can be used according to the present invention can also be produced by recombinant methods. This process is described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor, New York, Berger and Kimmel, M ethods in Enzymo 1 ogy, Vol. 152, Guide to Molecu-lar Cloning Techn ques (189 7), Academic Press Inc., San Diego CA, which has been included in this case for reference. Such recombinant antibodies can be produced in eukaryotic or prokaryotic cells by methods known in the art. Mammals, the paper size of the paper applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 520289 A7 B7 V. Description of the invention (I3) It is a lymphocyte strain. Best host cell. Chimeric, humanized or human antibodies are preferably produced recombinantly. Regions that can be selected for non-antigen-binding regions of antibodies are described in E.A. Kabat et al., Sequences of Proteins of Immunological Interest (1987), Nation-al Institute of Health, Bethesda MD. Recombinant antibody-L-selectin humanization and production of human antibodies are described in WO 94/1/2 2 15 and are also incorporated herein by reference. A particularly good humanized anti-L-selectin antibody is HuDreg 55, which is constructed in the same manner as the HuDr eg 200 described herein, and contains two light chains with the sequence of SEQ IN NO: 2 and two of them. SEQ IN NO: 4 The heavy chain of the sequence. Preferred humanized immunoglobulins are those that bind to the selectin with a binding affinity of at least 1 X 1 0 7 M-1 under standard binding conditions (standard conditions such as phosphate buffer at 25 ° C with 2% embryonic bovine serum added) Of salt water). Examples of such humanized immunoglobulins are HuDr eg 55 and HuDreg 200. A preferred humanized antibody is an affinity of at least 1 X 108 M-1 under standard binding conditions, and more preferably an affinity of at least 1 X 109 M-1, and even more beneficial is at least lxlO1. Binding agent with affinity above M-1 and human selectin. Usually the binding affinity of humanized immunoglobulins is within the 3-10 factor of the mouse immunoglobulins from which it is derived. For example, the affinity of the mouse Dr eg 200 antibody is about 108 M-1, and the mouse Dr eg 55 is about 109 M-10. The following examples, sequence strategies, publications, and pictures further clarify that the paper standard of this publication is applicable to Chinese national standards ( CNS) Α4 specification (210 × 297 mm) " ~ ~ 16-(Please read the precautions on the back before filling this page) Order 520289 A7 ___B7_ 5. Description of the invention (Η). It should be understood that the processes described are merely examples for illustration and are not limiting, others describe the subject matter of the present invention after modification. Example 1 Use of Hang-L-selectin antibody to reduce post-traumatic organ failure proves that humanized antibodies antagonize the protective effect of L-selectin (anti-L-selection.selectin) on reducing post-traumatic organ failure, as is common Occurs after injury in patients with severe multiple trauma. Humanized anti-L-selectin antibody (

Hu Dre e 55) acts as an antibody. It can also react with L-selectin of baboons. The mouse version of this antibody is described by Kishimoto in PNAS, USA 87 (1990) 2244-2248. The humanization sequence is shown in S E Q ID N 〇: 1 to 4 0 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page)

HuDreg 55 and HuDreg 200 antibodies can react with L-selectin on human white blood cells, but only HuDreg 5 5 can react with L-selectin on baboon white blood cells. Therefore HuDreg 55 was used. Because HuDreg 55 and HuDr eg 200 bind to human leukocytes at the same concentration range (as in the FACS analysis), most of the effects of HuDr eg 5 5 on baboons are comparable to those of HuDr eg 200. As for the pattern, inducers in baboons can have severe tissue damage (two inward and / or outward fluid and blood loss) with reduced blood volume. Pure blood loss plus subsequent shock (hemorrhagic shock) is less relevant to lung injury (Pretorius et al., J. Trauma 1987; 27: 1344-1353; Schlag et al., Page 384-402, in Schlag, Redl: This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) -17-520289 A7 __B7 _ V. Description of the invention (l5)

Pathophysiology of Shock, Sepsis, and Organ Failu-re, Springer Verlag, Berlin, 1 9 9 3). This is consistent with clinical experience that pulmonary complications are rarer than purely hemorrhagic shock (Sch lag et a 1 •, 193 supra). In order to determine the frequency and severity of post-traumatic lung failure, animals must be observed for several days (ie: SELIC 971, SELIC 9 7 9 (treatment group); and (: 0968, Co 969, Co 9 7 0 (control group)) ; Then, for moral reasons, fractures cannot be induced in unconscious animals and left untreated for several days, so that tissue trauma is simulated in this subchronic model. The activation of the complement system seems to be the earliest of the cellular system It starts and plays a key role in the rapid occurrence of non-bacterial inflammatory reactions in the body (Schlag et al., 193, shown above). Therefore, complement is activated by cobra venom factor in the model. In severe multiple The mortality rate for most post-traumatic organ failures is 15-30% in the relevant literature. In this animal model, the severity of multiple trauma increases to at least twice as high as the previous rate in humans. Therefore, The observation period is 3 days. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). Adult baboons with weights (BW) of 18 and 22 kg are in 3 Research is permitted after quarantine. Fasted animals are sedated with ketamine (6-8 mg / kg), then intubated and connected to a CPAP respirator (continuous positive tracheal pressure) (25 ± 2% inspiratory 〇2 Concentration). Maintain anesthesia with pentobarbital at 1.3 mg / kg / h. Animals can breathe spontaneously. Push the Swan Ganz catheter into the pulmonary artery and use the right jugular vein. The catheter for drawing blood and measuring blood pressure is tied to the right arm Arteries. Large lumen catheters are introduced into the neck. The paper size is applicable to Chinese National Standard (CNS) A4 (210X 297 mm) 520289 A7 _B7_ V. Description of Invention (I6) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please first Read the instructions on the back and fill in this page) to temporarily collect blood. Then introduce the blood transfusion, medical and blood collection catheter into the left arm vein. Bladder cannula to detect urine production. Swan Ganz catheter and arterial catheter are left for 3 days. For fluid balance, animals receive 5 ml / kg / hr Ringer's solution (an electrolyte solution for parenteral fluid replacement) during anesthesia. The temperature of the animal's blood is maintained at 237 ° C with the help of infrared lamps Perform blood gas analysis (P02, pC02, pH, BE, HC03-) and determine hemodynamic variables (MAP, RAP, PAP, CO, HR). Use respiratory rate (RR) and the last exhaled C 0 2 Determine lung function. Repeat blood collection to detect the number of white blood cells (WBCs). Cobra venom factor is administered at the beginning of retransfusion at a dose of i · ν.10 units / kg, and then administered 1 hour after retransfusion Dosage of 5 units / kg. Adjust the blood draw for the reduction of the starting blood volume so that the MAP (mean arterial pressure) can be between 40 and 50 mm Hg and the C0 (cardiac output) is reduced by 50-70% . About 50 ml / kg is usually drawn for this and stored until re-transfusion. Defective shrinkage is maintained for 2-3 hours and controlled in this way to keep its line beyond no more than 5 to 7mE Q. At the end of this shock period, re-transfusion of blood collected previously is started. This period lasts 4 hours. Ringer's solution was additionally administered to supplement re-transfusion. The humanized antibody Hu D r e g 5 5 or an equivalent volume of saline was used as a blank group 'and administered intravenously 15 minutes after the start of retransfusion. The anti-L-selectin antibody was administered at a dose of 2 mg / kg. At the end of the re-blood transfusion, the animal woke up from anesthesia and returned to the threshold for observation. At 24 hours, 48 hours, and 72 hours, low-level anesthesia was induced, and detection variables were registered and blood was drawn. This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) ^ 19-520289 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (Π) No death, sacrifice and post-mortem. The primary endpoints of the study were death, survival, and organ damage to the lungs. In the first experiment, three control animals were treated with a blank solution, and two were given HuD I · g 5 5 humanized antibodies. Of the three control animals, two died at 38 and 41 hours before the end of the three-day observation period, while two animals treated with anti-L-selectin survived. In the animals treated with antibodies, the degree of organ damage expressed by lung wet weight was almost normal (normal value 7-8 g / kg BW), while it was comparable in all three control animals treated with the blank group. Increase (Figure 1). This is due to the infiltration of fluids after osmotic disorders. In surviving animals, the cardiovascular variable C02 & MAP (Figures 2 and 3) was better than the control animals at 24 hours. The dying control animals also had a negative arterial baseline excess (BE), showing a chaotic acid-base balance (Figure 4). The increase in leukocytes (increased white blood cells) observed in control animals was not seen in antibody animals (Figure 5). Example 2 Use of anti-L-selectin antibodies to reduce post-traumatic mortality The experiment reported in Example 1 was continued and expanded to include 28 baboons, arbitrarily assigned to two experimental groups performed as described in Example 1 one. Baboons received 2 ml / kg of anti-L-selectin antibody, i.v., or a suitable blank volume-dose as a control group, which was injected 15 minutes after the start of reperfusion after the hemorrhage period. The primary endpoint of the study's statistical analysis was mortality and survival at the end of the 3-day observation period. Use Fisher's to confirm that the paper size is in accordance with Chinese National Standard (CNS) A4 specification (210X 297mm) (Please read the precautions on the back before filling out this page) Order-20-520289 A7 B7 V. Description of the invention (18) Field test Mortality analysis was performed, and log-rank-survival analysis was performed before the trial. Report unilateral P values (reduce mortality or prolong survival by active treatment). Only when the estimated statistical probability (P) < 0.05 is the non-useful hypothesis rejected. Anti-L-selectin antibody reduced mortality in the control group, from 10 of 14 baboons (= 70%) (ρ < 0.05) to 3 of the active treatment group (= 2 1%) It has statistical significance. In addition, the survival time in the anti-L-selectin group was extended to 64.4 hours, while the animals in the control group died earlier (P < 0.05), with an average of 42 hours. This difference is statistically significant. Condensed results ζ (please read the notes on the back before filling this page)

, 1T mortality, survival time (hours) anti-L-selectin antibody 3/14 " 64 · 4 土 4 · 7 + blank control group 10/14 42 · 4 土 5 · 7 Staff Consumer Cooperatives, Central Bureau of Standards, Ministry of Economic Affairs Standard deviation of printed average soil average; η = 14 (per group); *, ρ < 0 · 05 using Fisher's exact test; +, P < 0 · 0 5 using logarithmic one permutation-test. This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) -21-520289 A7 __B7 V. Description of the invention (19) The observation period is 72 hours. These data show that early treatment with anti-L-selectin for baboons with hemorrhagic-traumatic shock caused by hemorrhagic-traumatic shock can significantly prolong survival and reduce mortality, compared with the blank control group In terms of. '' Example 3 Use of anti-L-selectin antibodies to reduce organ damage after in vitro and liquid circulation studies Humanized antibodies against L-selectin, preferably Hu D reg 5 5, reduce organ damage after extracorporeal blood circulation Protective effect, this kind of situation often occurs after long-term operation of the lung machine in the heart surgery center. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) As for the mode, it can be operated for several hours in a baboon via a heart-lung machine, which can cause serious lung injury. Take over lung and heart functions after stopping. Once the machine is turned off, the pumping action of the heart is resumed, and the endogenous circulation and breathing action is resumed. Activated white blood cells will infiltrate the pulmonary circulation in large quantities and cause serious lung injury. The white blood cells present in the pulmonary circulation can locally release high concentrations of toxic mediators, which cause damage to the vascular endothelium and subsequent increase in permeability. During this process, fluid flows from the vascular space into the lung cells (the smallest lung cell), causing the fluid to accumulate in the lungs. This disrupts the exchange of gas in the lungs and therefore begins to depend on the population to breathe. Oxygen demand increases when fluoride exchange is severely increased, and because of the paper size of the lung cell-endothelial barrier, the Chinese National Standard (CNS) A4 (2K) x 297 mm is applicable.-22-520289 A7 B7____ V. Description of the invention (20) The fibroproliferative transformation effect will be worsened. Therefore, in a particularly severe case, the concentration of oxygen inhaled in the respiratory tract will increase from about 20% to about 100%. However, in this example, the supply of pure oxygen is not sufficient to maintain arterial oxygen concentration or partial pressure of oxygen at a moderate level. Fibroproliferative transformation and pulmonary edema increase pulmonary artery pressure, which is connected to the lungs and thus causes tension in the right ventricle. If these reactions accumulate, they will eventually die from cardiopulmonary failure. Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Make adult baboons with body weight (BW) between 18 and 22 kg to conduct research after 3 months of quarantine. The fasted animals were sedated with ketamine (6-8 mg / kg), intubated, and connected to a CP AP respirator (2 5 ± 2% inspiratory 02 concentration). Anesthesia was maintained with pentobarbital at 1 to 3 ml / kg / hour. Animals can breathe spontaneously. Then push the Swan Ganz catheter through the right jugular vein into the pulmonary artery. A catheter for drawing blood and detecting blood pressure was tied to the right arm artery. For transfusion, medical and blood collection catheters are introduced into the left vein. The bladder was intubated to measure urine output. To equilibrate the fluid, animals received 5 ml / kg / hour Ringer's solution. Animal blood temperature was maintained at 37 ° C with the help of infrared lamps. Blood gas analysis (P02, PC02, PH, BE, HC03-) was performed, and hemodynamic variables (MAP, RAP, PAP, Co, HR) were determined. Respiratory rate (RR) and the last exhaled CO2 are used to determine lung function. Blood was collected repeatedly to detect the number of white blood cells (W B C). The thorax was opened at the beginning of the experiment (thoracotomy), and the veins and aorta were prepared. After that, the venous cavity was first connected with the aorta with a cannula, so that blood from the venous cavity could flow into the cardiopulmonary machine, and then return to the aorta. 520289 A7 B7 5. Description of the invention (21). The peristaltic pump in the heart-lung machine performs the pumping function of the heart and ensures the pressure gradient required to maintain circulation. The exchange of oxygen and the combination of carbon dioxide are achieved by the action of membrane and oxygen. The blood is heparinized so that the pipes and blood vessels are not blocked. The blood returns to the aorta through the piping system and is distributed in the body through the normal vascular system. Cardiopulmonary machines take over the functions of the heart and lungs. The heart stops and the machine is in operation, so that the surgeon can work on the heart valve (insertion of an artificial object). Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). 15 minutes before the end of the extracorporeal circulation for 4 hours, dose 2 mg / kg HuD reg 5 5 or the same volume of the blank group Directly into the heart-lung machine system. Animals were observed for an additional 4 hours after the end of extracorporeal circulation. Repeat the previous test, before, during and after extracorporeal circulation. In particular, the variables of gas and acid-base balance in arterial blood, cardiovascular variables such as mean arterial blood pressure, right arterial pressure, pulmonary arterial pressure, cardiac output and heart rate, and pulmonary function (such as the last exhaled C 0 2), and Blood is drawn for hematology, clinical chemistry (such as kidney and liver function) and biochemical analysis. In addition, urine volume (renal function) was also measured. Furthermore, it also determines the variables of osmotic disorders in the lungs. Animals were sacrificed and necropsied at the end of the experiment, and histological examinations were performed to determine the degree of damage to various organs and systems, such as heart, lung, liver, kidney, intestine, CNS, blood, etc. Animals treated with HuDr eg 55 are expected to sustain less organ damage than the blank treatment group. Sequence Listing (1) General Information This paper size applies to Chinese National Standard (CNS) A4 specifications (210X297 mm)-24-520289 A7 B7 V. Description of invention (22) (i) Applicant (A) Name: Martin, Ulrich , et al. (ii) Invention name: anti-selectin antibody for preventing most organ failure after trauma and for preventing acute organ damage after extracorporeal blood circulation. (iii) Number of sequences: 4 (iv) Address: (A) Felfe & Lynch Attn: Norman D. Hanson (B) 805 Third Avenue (C) New York (D) New York (E) USA (F) 10022 (v) Computer readable type: (A) Media type: 3, 5 " Computer disk (B) Computer: IBM p C Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Compatible Economy (Please read the note on the back first) Please fill in this page again for matters) (C) Operating system: PC—DOS / MS—DOS (D) Software: ASCII (vi) Current application information (A) Application number: To specify (B) 20-DeC-95 (C) Category: 5 3 0 (vii) Previous application materials: (A) Application number: EP 95 1 1 2 8 9 5.8 (B) Date of filing: 17 — Aug — 1995 This paper size applies to Chinese National Standard (CNS) 8 4 Specifications (210 X 297 mm) 520289 • A 7 B7 V. Description of the Invention (23) (A) Application No .: EP 95 114 969.9 (B) Date of incorporation ·· 19-Sep-1995 (A) Application No. ·· US 08 5 7 8 9 5 3 (B) Date of filing: 27 — Dec — 1995 (viii) Legal affairs agency information (A) Name: Hans ο η, N 〇rman D · (B) Registration number: 30 , 946 (C) Reference / Index: BOER 1 0 5 9 -PFF / NDH (ix) Telex (A) Telephone: (212) 688-92 00 (B) Fax: (212) 838-3884 (2 ) SEQ ID NO: l Information (i) Sequence characteristics Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) (A) Length: 654 base pairs (B) Type: Nucleic acid (C) strand: double strand (D) topology: linear (ii) molecular type: cDNA (i X)

(A) Name / Index: CDS (B) Position: 1,654 (xi) Sequence description: SEQ ID Ν Ο: 1 This paper size applies to China National Standard (CNS) A4 specification (210 × 297 mm)-26- 520289 A7 ___B7 V. Description of the Invention (Μ) 4 4 4 2 9 1 ο 4 2 6

GAC ATT CAG ATG ACC CAA TCT CCG AGC TCT TTG TCT GCG TCT GTA GGG

Asp Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

GAT AGG GTC ACT ATC ACC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT

Asp Arg Val Thr lie Thr Cys Lys Ala Ser Gin Ser Val Asp Tyr Asp 20 25 30

GGT GAT AGT TAT ATG AAC TGG TAC CAA CAG AAA CCA GGA AAG GCA CCC

Gly Asp Ser Tyr Met Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro 35 40 45

AAG CTT CTC ATC TAT GCT GCA TCC AAC CTA GAA TCT GGT ATC CCA TCC

Lys Leu Leu Engineer Tyr Ala Ala Ser Asn Leu Glu Ser Gly lie Pro Ser 50 55 60

AGG TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC ACC ATC TCT

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser 65 70 75 80

TCT CTG CAG CCG GAG GAT TTC GCA ACC TAT TAC TGT CAG CAA AGT AAT

Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Asn 85 90 95

GAA GAT CCG TGG ACG TTC GGT CAA GGC ACC AAG GTG GAA ATC AAA CGA

Glu Asp Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Le Lys Arg 100 105 110 ACT GTG GCT GCA GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG 384-Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin I 115 120 125 、,,, and Ministry ^ TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT 43 2 Standard Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr f 130 135 140 Bureaux CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC CAA TCG 480 'Fee Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 145 150 155 160 Standards apply to China National Standards (CNS) A4 specifications (210X297 mm) -27-520289 A7 B7 V. Description of invention (25) GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC 528 Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr 165 170 175 TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAG GAG AAA 576 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180. 185 190 CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC 624 His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205 GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT 654 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 (Please read the notes on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (2) SEQ ID N0: 2 Data (i) Sequence characteristics (A) Length: 2 1 8 (B) Type: Amino acid (C) Share property: Single share (D ) Topology: Linear (ii) Molecular type: Protein (xi) Sequence description: SEQ ID NO: 2 This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) M9-28-520289 Central Standard of the Ministry of Economic Affairs Bureau's consumer cooperation, printed by the company A7 B7 V. Invention description (26)

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 l〇 15

Asp Arg Val Thr lie Thr Cys Lys Ala Ser Gin Ser Val Asp Tyr Asp 20 25 30 '

Gly Asp Ser Tyr Met Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro 35 40 45

Lys Leu Leu lie Tyr Ala Ala Ser Asn Leu Glu Ser Gly lie Pro Ser 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 65 70 75 80

Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Asn 85 90 95

Glu Asp Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg 100 105 110

Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Asp Glu Gin 115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 145 150 155 160

Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr 165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro 195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 This paper size is applicable to Chinese National Standard (CNS) M specification (210X297 mm) (Please read the precautions on the back before filling this page)

-29-520289 A7 B7 V. Description of Invention (27)

E S 2 / (V sequence \ Jy \ 1 / Nly XI / A B c D .1 (/ IN ((i

X

B

X A

B

X special style flutter color call color call Q line long stock extension special name special name order

Material 3 〇 N D 3 acid strand: 1 nuclear double: formula ...... type of base 9 2

A N D Type C S D C Index 9 2 3 (Please read the notes on the back before filling this page) Bowstring peptide t a m Q E S

D

3 ο N The Ministry of Economic Affairs, the Ministry of Economic Affairs, printed by the Consumer Cooperative Agency GAA GTG CAA CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCT GGA GGA 48

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 15 10 15 AGC TTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACT TTC AGT ACC TAT 96

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 This paper size applies to Chinese National Standard (CNS) A4 size (210x297 mm) -30-520289 A7 B7 V. Description of the invention (28) GCC ATG TCT TGG GTT CGC CAG GCT CCA GGG AAG GGA CTC GAG TGG GTC 144

Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Please read the notes and fill in this page 35 40 45 GCA TCC ATT AGT ACT GGT GGT AGC ACC TAC TAT CCA GAC AGT GTG AAG 192

Ala Ser lie Ser Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 GGC CGA TTC ACC ATC TCC AGA GAT AAT GCC AAG AAC ACC CTG TAC CTG 24 0

Gly Arg Phe Thr Engineering Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 CAA ATG AAT TCT CTG AGG GCT GAG GAC ACG GCC GTG TAT TAC TGT GCA 288

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys A'la 85 90 95 * AGA GAC TAT GAC GGG TAT TTT GAC TAC TGG GGC CAA GGC ACC CTG GTC 33 6

Arg Asp Tyr Asp Gly Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val 100 105 110 ACA GTC TCC TCA GCT TCC ACC AAG GGC CCA TCC GTC TTC CCC CTG GCG 384

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 CCC TGC TCC AGG AGC ACC TCC GAG AGC ACA GCC GCC CTG GGC TGC CTG 432

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC 480

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Employee Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Yin GCC CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA 528

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser 165 170 175 GGA CTC TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG 576 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 This paper size applies to Chinese National Standard (CNS) A4 size (21 × 29? Mm) ~ 31-520289 A7 B7 V. Description of invention (29) GGC ACG AAG ACC TAC ACC TGC AAC GTA GAT CAC AAG CCC AGC AAC ACC Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 AAG GTG GAC AAG AGA GTT GAG TCC AAA TAT GGT CCC CCA TGC CCA TCA Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser 210 215 220 TGC CCA GCA CCT GAG TTC CTG GGG GGA CCA TCA GTC TTC CTG TTC CCC Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 CCA AAA CCC AAG GAC ACT CTC ATG ATC TCC CGG ACC CCT GAG GTC ACG Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr 245 250 255 TGC GTG GTG GAC GTG AGC CAG GAA GAC CCC GAG GTC CAG TTC AAC Cys Val Val Val Asp Val Ser G in Glu Asp Pro Glu Val Gin Phe Asn 260 265 270 TGG TAC GTG GAT GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 GAG GAG CAG TTC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 CTG CAC CAG GAC TGG CTG AAC GGC AAG GAG TAC AAG TGC AAG GTC TCC Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 AAC AAA GGC CTC CCG TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC AAA Asn Lys Gly Leu Pro Ser Ser lie Glu Lys Thr lie Ser Lys Ala Lys 325 330 335 GGG CAG CCC CGA GAG CCA CAG GTG TAC ACC CTG CCC CCA TCC CAG GAG Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu 340 345 350 GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 This paper size applies to China National Standard (CNS) A4 specifications (210X 297 mm) (Please read the notes on the back first Refill this page) Order 912 960 «1008 1056 1104 520289 A7 B7 V. Description of the invention (3〇) TAC CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG Tyr Pro Ser Asp Worker Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu 370 375 380 AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 TTC CTC TAC AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG CAG GAG GGG Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly 405 410 415 1152 7 Please read the precautions on the back 1200 and 1248 before

AAT GTC TTC TCA TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC 1296 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 ACA CAG AAG AGC CTC TCC CTG TCT CTG GGT AAA Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 1329 Order printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (2) SEQ ID NO: 4 Data (i) Sequence characteristics: (A) Length: 4 4 3 (B) Type: Amino acid (C) strand property: double strand (D) topology: linear (ii) molecular type · protein (X i) sequence description: s EQ ID Ν Ο: 4 This paper size applies to Chinese National Standards (CNS) A4 specifications (297 mm> MW. -33-520289 A7 B7 V. Description of the invention (31)

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Gly Leu Val Gin Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr |

20 25 30 I

Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Ser Gong Ser Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60

Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala; 85 90 95 i

Arg Asp Tyr Asp Gly Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val 100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125,

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Please read the notes on the back before filling out this page)

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser 165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser 210 215 220 This paper size applies to the Chinese National Standard (CNS) A4 size (210X 297 mm)-34-Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 520289 A7 B7 V. Description of the invention (32)

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Engineer Ser Arg Thr Pro Glu Val Thr 245 250 255

Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn 260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285

Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300

Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320

Asn Lys Gly Leu Pro Ser Ser lie Glu Lys Thr lie Ser Lys Ala Lys 325 330 335

Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu 340 345 350

Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Ph ^ 355 360 365

Tyr Pro Ser Asp Tool Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu 370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly 405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430

Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 This paper size is applicable to China National Standard (CNS) A4 size (210X 297 mm) (Please read the precautions on the back before filling this page)

Order

Claims (1)

520289 A8 B8 C8 D8 VI. Scope of Patent Application Annex I (A) (Please read the notes on the back before filling out this page) No. 8 5 1 1 2 85 Chinese Patent Application Scope Amendment 12 Month; 2nd amendment 1 * A pharmaceutical composition for treating multiple traumatic events, which contains a therapeutically effective dose of an anti-selectin antibody and a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to item 1 of the application, wherein the anti-selectin antibody is an anti-L-selectin antibody. 3. The pharmaceutical composition according to item 1 of the patent application scope, wherein the anti-selectin antibody is an anti-E-selectin antibody. 4. The pharmaceutical composition according to item 1 of the application, wherein the anti-selectin antibody is an anti-P-selectin antibody. 5. The pharmaceutical composition according to item 1 of the patent application scope, which contains 1.0 to 10 mg of anti-selectin antibody per kilogram of body weight. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 6 · If the pharmaceutical composition in the scope of patent application No. 1 is applied, it is administered 0.5 to 8 hours after the multiple traumatic event. 7. The pharmaceutical composition according to item 6 of the patent application, which is administered 0.5 to 4 hours after a multiple trauma event. 8. The pharmaceutical composition according to item 5 of the patent application, which is administered at intervals of 6 and 72 hours. 9. The pharmaceutical composition according to item 8 of the patent application, which is administered at intervals of 6 and 36 hours. This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 1-520289 A8 B8 C8 D8 VI. Scope of patent application (please read the notes on the back before filling this page) 1 〇 · If the scope of patent application The pharmaceutical composition of item 1 is administered up to 10 days after a multiple trauma event, and the dosage and time of the administration are from the previous dose of anti-selectin antibody in the serum or plasma of the patient at an interval of 6 to 24 hours after the previous dose. The concentration is determined, wherein when (a) the concentration of the anti-selectin antibody is lower than 10 micrograms per milliliter of the patient's serum or plasma, the dose is at least as high as the previous dose, and can be as high as 10 mg / The dose in kilograms, or when (b) the anti-selectin antibody concentration is between 10 micrograms and 50 micrograms per milliliter of the patient's serum or plasma, half the previous dose is administered, or when, (c ) When the concentration of the anti-selectin antibody is greater than 50 micrograms per milliliter of the patient's serum or plasma, the anti-selectin antibody and the pharmaceutically acceptable carrier are not administered, and further according to steps (a) and (b) Track patient's serum or blood . 1 1 · The pharmaceutical composition according to item 1 of the application, wherein the anti-selectin antibody is a humanized antibody. 1 2 · The pharmaceutical composition according to item 11 of the scope of patent application, where the humanized antibody printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is an anti-L-selectin antibody HuD reg 5 5 or \ Hu D r eg 200. 1 3-A pharmaceutical composition that reduces the likelihood of the occurrence of multiple post-traumatic organ failure, comprising a therapeutically effective dose of at least one anti-selectin antibody in combination with a pharmaceutically acceptable carrier. 1 4 · The pharmaceutical composition according to item 13 of the application, wherein the anti-selectin antibody is humanized. This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) 1 2-520289 A8 B8 C8 D8 VI. Application scope of patents 15. For the pharmaceutical composition No. 13 of the scope of patent application, where the anti- Selectin antibodies are anti-L-selectin antibodies. 16. The pharmaceutical composition according to item 13 of the application, wherein the anti-selectin antibody is an anti-P-selectin antibody. 17. The pharmaceutical composition according to item 13 of the patent application scope, wherein the anti-selectin antibody is an anti-L-selectin antibody. 18. The pharmaceutical composition according to item 13 of the patent application, wherein the anti-L-selectin antibody is Dr eg 55 or HuDr eg 5 5 ° 1 9. The pharmaceutical composition according to item 13 of the patent application Among them, the organ failure is most organ failure. (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs This paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -3-