CN1188528C - Intercellular adhesion molecule-3 and its binding ligands - Google Patents

Abstract

The present invention relates to intercellular adhesion molecules (ICAM-3) which is involved in the process through which lymphocytes recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention is directed toward such molecules, screening assays for identifying such molecules and antibodies capable of binding such molecules. The invention also includes therapeutic and diagnostic uses for adhesion molecules and for the antibodies that are capable of binding them.

Description

ICAM-3 and part thereof

The application relates to U.S. Patent application series number 07/045,963 (Mays 4 1987 applying date), 07/115,798 (November 2 1987 applying date), 07/155,943 (the applyings date, on February 16th, 1988), 07/189,815 (Mays 3 1988 applying date), 07/250,446 (September 28 1988 applying date), 07/454,294 (December 22 1989 applying date) and PCT application number PCT/ US91/02942 and PCT/US91/02946 (applying date of accepting office in the U.S. is on April 27th, 1991), this paper list of references is all classified in these applications as.

The present invention relates to a kind of new ICAIU of naming as ICAM-3, this molecule is participated in the identification lymphocyte population and is adhered to process on the cellular matrix. ICAM-3 transmitting inflammation positions and the lymphocyte at immune position and the cell interaction of macrophage.

The invention still further relates to independent use ICAM-3 or unite use with ICAM-1 and/or ICAM-2, to suppress the intercellular adhesion of granulocyte, lymphocyte or macrophage pedigree. Use above-mentioned molecule that a kind of method for the treatment of specificity and nonspecific inflammation is provided.

The invention still further relates to independent use ICAM-3 or unite use ICAM-1 and/or ICAM-2 suppress HIV particularly HIV-1 to lymphocytic infection, the method that has contacted HIV or infected the individuality of HIV with treatment or prevention. Thereby it can be used for treating such as diseases such as the AIDS that is caused by HIV virus (aids).

The invention still further relates to independent use ICAM-3, or unite and use dead and " plasomidum " formation of ICAM-3 and ICAM-1 and/or ICAM-2 suppressor T cell, to treat by the individual method of HIV infection. Therefore it can be used for treating disease, such as the AIDS (aids) that is caused by HIV-1 virus.

The present invention relates to use separately ICAM-3, or unite and use ICAM-3 and ICAM-1 and/or ICAM-2 treatment asthma.

In addition, the invention still further relates to the molecule (hereinafter referred to as anti-ICAM-3) that to be combined with ICAM-3. Anti-ICAM-3 molecule is intended to regulate the biological function relevant with ICAM-3 with the ICAM-3 combination. Binding molecule of the present invention can be antibody, peptide or the carbohydrate that can be combined with ICAM-3. These binding molecules can be used for regulating the biological function of ICAM-3.

The invention still further relates to the anti-ICAM-3 of independent use, or unite and use anti-ICAM-3 and anti-ICAM-1 and/or anti-ICAM-2 to suppress the intercellular adhesion of granulocyte, lymphocyte or macrophage pedigree. Use these molecules that the method for the treatment of specificity or nonspecific inflammation is provided.

The invention still further relates to by the anti-ICAM-3 of independent use or unite and use anti-ICAM-3 and anti-ICAM-1 and/or anti-ICAM-2, the individual endolymph cell that suppresses to have contacted HIV is by the HIV treatment and the prevention method that infect of HIV-1 particularly. Therefore it can be used for treating disease, such as the AIDS (aids) that is caused by HIV virus.

The invention still further relates to the anti-ICAM-3 of independent use or unite and use anti-ICAM-3 and anti-ICAM-1 and/or ICAM-2, to suppress cell that HIV-1 infects by the methods for the treatment of of moving in the circulatory system. Therefore it can be used for treating disease, such as the AIDS (aids) that is caused by HIV-1 virus.

The invention further relates to the anti-ICAM-3 of independent use, or unite and use anti-ICAM-3 and anti-ICAM-1 and/or anti-ICAM-2 treatment asthma.

A. leucocyte adheres to and function

In order to make the host suitably resist the intrusion of the external pathogenic factors such as bacterium or virus, leucocyte must can be attached on the cellular matrix (referring to Eisen, H.W., Microbiology, 3rd Ed., Harper ﹠ Row, Philadelphia, PA (1980), pp.290-295 and 381-418). Leucocyte must be able to be attached on the endothelial cell could move to inflammation part from circulation. In addition, they must be attached on the antigen presentation cell normal specific immune response could occur. At last, they must be attached to and could dissolve cell or the tumour cell that is subject to virus infections on the suitable target cell.

Recently, used hybridoma technology to identify to participate in the leukocyte surface molecule of the function of the above-mentioned adhewsive action mediation of mediation. Briefly, identified anti-human T cell (Davignon, D.et al., Proc.Natl.Acad.Sci.USA 78:4535-4539 (1981)) and mouse boosting cell (Springer, T.et al., Eur.J.Immunol.9:301-306 (1979)) monoclonal antibody, these monoclonal antibodies can be incorporated into leukocyte surface and suppress above-mentioned adhesion correlation function (Springer, T.et al., Fed.Proc.44:2660-2663 (1985)). Molecule by these Identification of the antibodies is called as Mac-1 and leucocyte function-associated antigen-1 (LFA-1). Mac-1 is the heterodimer that sees on macrophage, granulocyte and the large granular lymphocyte. LFA-1 is the heterodimer (Springer, T.A.et al., Immunol.Rev.68:111-135 (1982)) that sees on most of lymphocytes. These two kinds of molecules add the third molecule, i.e. p150.95 (it has the tissue similar to Mac-1 and distributes), and (Keizer, G.et al., Eur.J.Immunol., 15:1142-1147 (1985)) all works in cell adherence.

Found that above-mentioned leucocyte molecule is member (Sunchez-Madrid, F.et al., the J.Exper.Med.158:1785-1803 (1983) of the relevant family of glycoprotein; Keizer, G.D.et al., Eur.J.Immunol.15:1142-1147 (1985)), be called " CD-18 family " of glycoprotein. This glycoprotein family is comprised of the heterodimer with a α chain and a β chain. Although the α chain between each antigen differs from one another, the β chain is high conservative (Sanchez-Madrid, F.et al., J.Exper.Med.158:1785-1803 (1983)). The β chain molecular weight of finding glycoprotein family is 95kd, and the α chain molecular weight does not then wait (Springer, T., Fed.Proc.44:2660-2663 (1983)) by 150 to 180kd. Although the α subunit of memebrane protein is not shared by the total extension homology of β subunit, recently the analysis showed that of glycoprotein α subunit is had substantial similitude between them. Sanchez-Madrid, the people such as F. (J.Exper.Med.158:586-602 (1983); J.Exper. Med.158:1785-1803 (1983)) α of the relevant glycoprotein of IFA-1 and the similitude between the β subunit have been commented.

Identified one group of individuality (Anderson, D.C.et al., Fed.Proc.44:2671-2677 (1985) that can not express with positive constant any member of this attachment proteins family at its leukocyte surface; Anderson, D.C.et al., J.Infect.Dis.152:668-689 (1985)). These individualities be thought suffering from " leukocyte adhesion deficiency disease " (" LAD ") (Anderson, D.C., et al., Fed.Proc.44:2671-2677 (1985); Anderson, D.C., et al., J.Infect.Dis.152:668-689 (1985)). Patient's LAD characteristic performance comprises that soft tissue injury, vomica form and wound is difficult for healing, and external adhesion dependence leukocyte function is unusual and infect responsive to chronic and recidivity bacterium. The granulocytic external behavior and their the corresponding normal control cell that derive from these patients LAD have same defective performance when anti-CD18 monoclonal antibody exists. That is to say that they can not be carried out and gather or be attached to the first-class adhesion correlation function of endothelial cell. But the more important thing is, observe these patients and can not produce normal inflammatory reaction because its granulocyte is not attached to the ability on the cellular matrix. The most significant is to observe because these patients' LAD granulocyte is not attached near the ability on the vascular endothelial cell of inflammation damage location, so can not reach inflammation part such as skin infection position. This adhering to is the steps necessary that infiltrates.

The external defective of these patients' lymphocyte performance is similar by the defective of the normal corresponding cell of antibody antagonism with those CD-18 molecule families. In addition, owing to these individual granulocytes can not adhere on the cellular matrix, so they do not have ability to produce normal immunoreaction (Anderson, D.C.et al., Fed Proc.44:2671-2677 (1985); Anderson, D.C.et al., J.Infect.Dis.152:668-689 (1985)). These data show that when lymphocyte can not adhere to normal mode owing to lacking the functional adhesion molecule of CD-18 family, i.e. performance has slowed down immune response.

In a word, leucocyte is for keeping animal health and survival ability, and they must stick on other cells (such as endothelial cell). Found the cell that this adhesive attraction need to have the specificity acceptor molecule on the leukocyte surface and participate in-cell contact. These are subjected to physical efficiency to make leukocyte adhesion on other leucocytes, endothelial cell and other non-vascular cells. Found LFA-1, Mac-1 and p150,95 molecules such as cell surface receptor such as grade are height correlations to each other. The people that leucocyte lacks these cell surface receptors easily suffers from chronic and the recidivity infection, and other Comprehensive Clinical diseases that comprise the defective antibody response.

In addition, because leukocyte adhesion has been participated in identification and repelled the process of external organization, in parenchymatous organ's (such as kidney) transplanting, unsubstantiality organ (such as marrow, tissue) transplanting, allergy and oncology field, important value is arranged so understand this process in depth.

It is the cause of disease of AIDS that HIV infects. Described many HIV mutation: two main mutation are HIV-1 and HIV-2. HIV-1 is popular in North America and Europe, and HIV-2 is popular in the Asia. Virus has similar structure, and coding has the protein of identity function. It is believed that it is to be attached to the upper (Schnittman that occurs of acceptor molecule (being called " CD4 ") that is present on T4 (" T the is auxiliary ") lymphocytic cell surface by virus protein (being called " gp120 ") that HIV infects, S.M.et al., J.Immunol. 141:4181-4186 (1988), the document is classified this paper list of references as). After being attached on this receptor, virus namely enters cell and copies, and kills the T cell in this process. Therefore the destruction of the T4 cell mass of individuality is the direct result that HIV infects.

The ability that the destruction of T cell makes infected patient resist opportunistic infect suffers damage. Usually become the tumour patient although infected the people of being grown disease, the relation between these tumours and HIV infect in most of the cases it be unclear that.

Although HIV virus copy for infected cell to be deadly property, only in the infected person's of sub-fraction T4 cell, to detect this copying. Nearest result of study prompting, the incidence of viremia virusemia is than the height of estimating in the past, and T cell infection frequency can be up to 1%.

Other mechanism that the virus-mediated T4 of HIV colony destroys have been illustrated in several research work.

Except HIV copies, also can destroy the cell that infected by HIV by the cellular toxicity killer cell. Killer cell generally all is present in the human body, plays the effect that monitors the host and destroy the external cell that may run into (such as the blood transfusion of mispairing or organ transplant etc.). After infected by HIV, the T cell presents the gp120 molecule at its cell surface. Killer cell is identified as external cell (rather than intrinsic cell) with these T4 cells, and then destroys it.

HIV infects the destruction that also can cause the healthy cell of uninfection. Infected cell can be secreted HIV-1 gp120 in hematological system. Then free gp120 molecule just can be attached on the CD4 acceptor of healthy cell of uninfection. This combination can make cell present the barment tag of the cell of HIV infection. The cellular toxicity killer cell is identified the gp120 on the T4 cell that has been combined in not infection, thus think that this cell is external, and the destruction of mediated cell.

Also have one for the useful especially mechanism of the present invention, in this mechanism, HIV forms by syncytial cell to cause the T4 cell death. " syncytial cell " is the giant cell that a plurality of nuclears are arranged, and merges and forms by reaching a hundreds of T4 cell. After the infected by HIV so that infected cell has obtained the ability that merges mutually with other T4 cells (comprising HIV healthy cell that infect or uninfection). The syncytial cell function of can not bringing into normal play, and can very fast death, thereby the destruction that causes T4 cell that HIV infects and that do not infect. This process is that the present invention is interested especially, because it has caused the intercellular direct cell of T4-cell contact. The cell of HIV infection forms plasmodial ability and shows that these cells need to have a kind of means that merge with healthy cell.

As if HIV infects, and particularly HIV-1 infects, affect the cell surface expression of leucocyte integrin (integrins), and by the cell adherence reaction (Petit of these heterodimers mediation, A.J., et al, J.Clin.Invest.79:188 (1987); Hildreth, J.E.K., et al., Science 244:1075 (1989); Valentin, A., et al., J.Immunology 144:934-937 (1990); Rossen., R.D., et al., Trans.Assoc.American Physicians 102:117-130 (1989), all these documents are all classified this paper list of references as). After infected by HIV-1, increased the homotypic aggregation (Petit, A.J., et al., J.Clin.Invest.79:188 (1987)) of U937 cell because of the cell surface expression of CD18 and CD11b. The U937 cell that HIV-1 infects adheres on the endothelial cell of IL-1 stimulation with the U937 cell than uninfection with larger frequency; This behavior can process infected cell with anti-CD18 or anti-CD11a monoclonal antibody or with anti-1CAM-1 antibody treatment endothelial cell matrix after be suppressed (Rossen, R.D., et al., Trans.Assoc. American Physicians 102:117-130 (1989)) confirmed. Found that also anti-CD18 or CD11a monoclonal antibody can suppress to relate to the lymphoblast of lectins (PHA) stimulation and the Syncytium formation (Hildreth of the negative T cell of infected in fact CD4, J.E.K., et al., Science 244:1075 (1989)). Found to only have the virus infected cell of processing with anti-CD18 or anti-CD11a monoclonal antibody on almost not impact of Syncytium formation, this point points out these antibody basically can protect the target cell that does not infect to avoid infecting (Hildreth, J.E.K.et al., Science 244:1075 (1989); Valentin, A.et al., J.Immunology 144:934-937 (1990)). Recently, the people such as Valentin (J.Immunology 144:934-937 (1990)) have further confirmed above-mentioned observed result, find that the monoclonal antibody special to CD18 can suppress Syncytium formation when continuous T clone and HIV-1 infection U937 cell are cultivated altogether.

Although the mechanism that the cell that CD18 or CD11a specificity monoclonal antibody protection sensitive cells avoid infecting with HIV occurs to merge it be unclear that; and Related Mechanisms the present invention neither be necessary for estimating; but point out with the research that radiolabeled gp120 does; the heterodimer that contains CD18 does not provide the binding site (Valentin of virus; A.; et al., J.Immunology 144:934-937 (1990). Therefore, HIV infects and has comprised cell-cell interaction, and/or simulates the virus-cell interaction of this cell-cell interaction. Cell-cell interaction can cause the transportation of acellular virus or the cell of virus infections to pass through the endothelial barrier conveying. Virus-the cell interaction of analog cell-cell interaction can be conducive to free virus is adhered to and/or infect healthy cell.

Therefore, the present invention is based on to a certain extent HIV and infects HIV-1 particularly and infect this observed result of expression that can improve CD11a/CD18 heterodimer and binding partner thereof. The raising of this expression is highly significant, because it has strengthened the ability (" homotypic aggregation " namely occurs) that T cell that HIV infects is adhering to each other or assemble. Because between static normal leucocyte, there are no this homotypic aggregation, be that this gathering of generation is necessary so this discovery shows the expression of CD11/CD18 acceptor and/or part such as ICAM-1. For homotypic aggregation occurs, LFA-1 must be attached on the ICAM-1. As described herein, ICAM-3 just on static T cell with the member of the ICAM molecule family of high level expression. Unless the T cell is " being activated ", otherwise only have anti-ICAM-3 antibody can the adhesion of blocking t cell on LFA-1. Therefore, anti-ICAM-3 antibody can be used for the gathering of suppressor T cell.

In addition, anti-ICAM-3 antibody can be used for blocking the adhesion process of infected T cell, and then allows HIV-1 to be relayed to patient's healthy cell by infected cell, also allows or be conducive to free virus to infect healthy cell.

The migration of the cell of C.HIV infection

It is very important that leukocytic migration and distribution avoid further infecting for the protection individuality. Yet these processes also cause by leukocytic migration and the distribution of virus infections. Noticeable especially leukocytic migration and the distribution infected by HIV. The migration of these cells causes the formation of the outer focus of blood vessel, and can cause that tumour and other are unusual.

The histological examination of damaged organ shows the outer monocytic infiltration of local vascular. Attempt to identify the virus infected cell that infiltrates in the central nervous system, found that the cell that exists HIV-1 to infect in the infiltrate. These studies show that HIV-1 virus mainly is present in monocyte and the macrophage, and (R.T.Johnson, et al., FASEB J., 2:2970 (1988) in other cells of this pedigree; M.H.Stoler et al, J.Amer, Med.Assn., 256:2360 (1980); S.Gartner et al., Science 233:215 (1986)).

Also do not determine in the past to stimulate the monocytoid cell of HIV-1 infection to form the outer mechanism that infiltrates of blood vessel. This mechanism may relate to the transportation of free virus, or viral transportation of passing endothelial barrier in infected monocyte endochylema.

Because the cell surface expression of HIV-1 infection stimulation molecule is conducive to leucocyte and adheres at vascular endothelial cell, and be conducive to leucocyte and from blood vessel, transfer to extravascular tissue position (C.W.Smith et al., J.Clin.Invest.82:1746 (1988), list this paper list of references in), so existing people proposes to prevent with the antibody that suppresses cell migration the diffusion (WO90/13316) of the cell of HIV infection.

D. asthma: Clinical symptoms

Asthma is the disease family of an allos, it is characterized in that bronchus has high response (Kay, A.B., Allergy and Inflammation, Academic Press, NY (1987) to stimulus; This article has been classified this paper list of references as). Clinically, as seen there is bronchus generally narrow, the secretion of thickness is arranged, patient's signs such as sound that paroxysmal dyspnea occurs, cough and stridulate. Although also do not know the relative consequence of these symptom, the long and is that the ventilation resistance increases lung and chest high level expansion, and the unusual distribution of ventilation and lung blood flow. Between this disease shows between asymptomatic stage or acute stage symptom occurs. Acute stage, can occur for hypoxgia and can cause death.

Whole world crowd has 3% to suffer from asthma approximately.

Two types asthma has been described: allergic asthma and atopic asthma. Allergic asthma is usually relevant with heritable allergic disease such as rhinitis, nettle rash, eczema etc. Be characterised in that to occur hypodermic air borne antigen (such as pollen, environment and the professional pollutant etc.) wheal-flare reaction that is negative, and serum IgE level occur and increase. In the cause of disease and exist IgE antibody relevant as if many patients develop into allergic asthma. Performance does not have the asthma patient of above-mentioned feature to be considered to suffer from atopic asthma.

It is believed that sex change asthma depends on IgE reaction, the latter is by T and bone-marrow-derived lymphocyte control, and is activated by air borne antigen and interaction in conjunction with the pre-formed IgE molecule of mast cell. For making individual sensitization, antigen must gather for a long time so that reach the concentration that can cause IgE to produce completely. In case sensitization, asthma patient can symptom occur in the reaction to extremely low antigen levels.

Asthma can trigger antigen, environmental factor, occupational factor, manual labor and the factor such as excited increases the weight of because of capacity.

Can treat by antagonist (such as atropine) with methyl xanthine (such as theophylline), Beta-3 adrenergic activator (such as catecholamine, resorcinol, saligenin and ephedrine etc.), glucocorticoid (such as hydrocortisone), mast cell degranulation inhibitor (being chromone, such as nasmil) and choline.

It is believed that asthma has comprised eosinophil flow in the lung tissue (J.Allergy Clin.Immunol.77:527-537 (1986), the document is classified this paper list of references as for Frigas, E.et al.).

According to bronchoalveolar lavage research (Godard, P.et al., J.Allergy Clin.Immunol.70:88 (1982)) result and to the research (Flavahan of the airway smooth muscle tissue of peelling off epithelial cell, N.A.et al., J.Appl.Physiol.58:834 (1985); Barnes, P.J.et al., Br.J.Pharmacol.86:685 (1985)) result understood the amynologic basis of asthma in depth. Although the amynologic mechanism that these researchs are not finally illustrated asthma, but they have developed hypothesis that the general acceptable immune cause that relates to this disease learns (referring to Frigas, E.et al., J.Allergy Clin.Immunol. 77:527-537 (1986)).

The Pathologic Characteristics of asthma be eosinophil to a large amount of infiltrations of pulmonary parenchyma, and the infringement of mucomembranous cilium capacity. " eosinophil hypothesis " prompting eosinophil attracted to the harmful mediators that is discharged by pulmonary mastocyte with neutralization on the bronchus. According to this hypothesis, eosinophil attracted on the bronchus, takes off particle here to discharge the cellular toxicity molecule. After taking off particle, eosinophil discharges enzyme, such as histaminase, aryl sulfatase and phosphatidase D in order in the enzymatic and harmful mediators of mast cell. Yet these molecules also promote the mucomembranous cilium disorganization, thereby have hindered the removing of bronchial secretion and cause the injury of lungs characteristic of asthma to change.

Because asthma relates to the migration of cell, so there is the people to propose to alleviate allergen to patient's impact (WO90/10453) with the antibody that suppresses this migration.

The present invention is based on the new cell adhesion molecule of having found to be called ICAIU 3 (ICAM-3). The invention still further relates to the functional derivatives of ICAM-3, anti-ICAM-3 antibody, become the fragment for the said antibody of the anti-ICAM-3 antibody of people, and can in conjunction with and suppress other molecules of the biological function of ICAM-3.

The present invention also comprises diagnosis and the treatment application of all above-mentioned molecules.

Specifically, the present invention includes ICAM-3 or its functional derivatives that there is no natural pollutant.

The invention provides acquisition and can encode or express the method for the molecule of the restructuring of ICAM-3 or its functional derivatives or synthetic DNA.

The present invention also provides the antibody, particularly monoclonal antibody that can be attached on the molecule that is selected from ICAM-3 and functional derivatives thereof.

The present invention also provides the hybridoma that can produce above-mentioned monoclonal antibody.

The present invention includes the method that produces required hybridoma, said hybridoma can produce the antibody that can be combined with ICAM-3 or its functional derivatives, and the method comprises the following steps:

A) be selected from pure in fact ICAM-3, express ICAM-3 cell, express the cell of ICAM-3 film, with the immunogene immune animal of the fragments of peptides of the fragments of peptides of carrier-bound ICAM-3, ICAM-3 or the ICAM-3 of being combined with carrier,

Splenocyte and the myeloma cell that b) will separate in the said immunized animal body are merged,

C) make the spleen that merged and myeloma cell form can secretory antibody the myeloma cell,

D) screening can produce the hybridoma of anti-ICAM-3 antibody.

The present invention also provides the method for the cell biological function of regulating the ICAM-3 mediation, said method comprises the ICAM-3 conditioning agent that uses effective dose to the object of this processing of needs, and wherein said ICAM-3 conditioning agent is selected from the fragment that can be combined with ICAM-3 of the antibody that can be combined with ICAM-3, said antibody, functional derivatives and the NIg antagonist except ICAM-1, ICAM-2 or CD-18 molecule family member of ICAM-3, ICAM-3.

The present invention also provides the method for the treatment of people and other mammiferous specificity inflammation, said method comprises that the object to this treatment of needs uses the antiinflammatory that can suppress the inflammation amount completely, wherein antiinflammatory is selected from the antibody that can be combined with ICAM-3, the fragment that can be combined with ICAM-3 of said antibody, the NIg antagonist of pure in fact ICAM-3, the functional deriv of ICAM-3 or ICAM-3, wherein said inflammation is caused by parenchymatous organ's transplanting (such as kidney), unsubstantiality organ transplant (such as marrow) or tissue transplantation.

The present invention also provides the method for the treatment of people and other mammiferous nonspecific inflammations, said method comprises to the object of this treatment of needs uses the antiinflammatory that is enough to suppress the inflammation amount, and wherein antiinflammatory is selected from the fragment that can be combined with ICAM-3 of the antibody that can be combined with ICAM-3, said antibody, the basically functional derivatives of pure ICAM-3, ICAM-3 or the NIg antagonist of ICAM-3.

The present invention further comprises the method for above-mentioned treatment inflammation, and wherein inflammation is with to be selected from each following pathological conditions relevant: adult's respiratory distress syndrome; Be secondary to multiple organ injury's syndrome of septicemia, hemorrhage or wound; The reperfusion injury of cardiac muscle or its hetero-organization; Acute glomerulonephritis; Adjuvant arthritis; With acutely inflamed skin disease; Acute purulent meningitis or other inflammatory disease of central nervous system, such as apoplexy: heat waste is anti-: haemodialysis: the special-shaped disease of leucocyte: ulcerative colitis: Crohn's disease: NEC: the toxicity that granulocyte infusion related syndrome and cytokine are induced.

The present invention comprises that also suppressing the hematopoiesis tumour cell (must utilize the member of CD-18 during this cell migration, LFA-1 particularly) method that shifts, said method comprises that the patient to this treatment of needs provides the preparation that can suppress above-mentioned transfer amount completely, and wherein said preparation is selected from the NIg antagonist of toxin relevant derive product or the ICAM-3 except CD-18 molecule family member of the functional derivatives of the derivative of the fragment that can be combined with ICAM-3, said fragment and toxin that can relevant derive product, said antibody with the toxin of the antibody of ICAM-3 combination, said antibody, the functional derivatives of ICAM-3, said ICAM-3.

The present invention also comprises the method for the tumour cell growth that suppresses expression ICAM-3, said method comprises that the patient to this treatment of needs provides the preparation that can suppress to grow completely, wherein said preparation is selected from the antibody that can be combined with ICAM-3, the functional derivatives that the product of deriving of said antibody and the fragment that can be combined with ICAM-3 of the product of deriving of toxin, said antibody, said fragment and toxin, member that the toxin of CD-18 molecule family is derived or CD-18 molecule family members' toxin is derived.

The present invention also provides and has detected the method that whether has the cell of expressing ICAM-3, and said method comprises:

A) can with cultured cell or cell extract in the presence of the nucleic acid molecules of ICAM-3 mRNA hybridization, and

B) detect and the nucleic acid molecules that is present in the complementary nucleic acid molecule hybridization in said cell or the cell extract.

The present invention also provides the method that whether has ICAM-3 in the detection of biological humoral sample, and the method comprises:

A) use and can be incubated with said sample with antibody or its fragment of ICAM-3 combination, and

B) detect said antibody and whether combine said sample.

The present invention also provides the pharmaceutical composition that comprises following composition:

A) be selected from following one group preparation: the antibody that can be combined with ICAM-3; The fragment that can be combined with ICAM-3 of said antibody, pure ICAM-3 basically, the functional derivatives of ICAM-3, or the NIg antagonist of the ICAM-3 except CD-18 molecule family member; And b) a kind of immunodepressant.

Fig. 1 shows that clone (A) and lymphocyte (B) are to the adhesion by the LFA-1 of the purifying of ICAM-1, ICAM-2 and ICAM-3 representative. (A) in the presence of the sealing MAb special to IFA-1, ICAM-1, ICAM-2 and ICAM-3, the cell of BCECF mark is combined on the coated microtiter well of IFA-1. Control wells is not coated with LFA-1.

Fig. 2 shows the flow cell analysis of accounts result to ICAM-1, the ICAM-2 of cell and ICAM-3 expression. (A) with MAb RR1/ 1 (anti-ICAM-1), MAb CB-IC2/1 (anti-ICAM-2), MAb CBR-IC3/1 (anti-ICAM-3) or uncombined contrast MAb X63 (fine rule) the mark lymph matricyte system of saturation capacity, then add the anti-mouse immuning ball protein of FITC. (B) analyzing as stated above 3 days lymphocytic ICAM of static people's lymphocyte and PHA activation expresses.

Fig. 3 shows the immunoprecipitation of ICAM-3. (A) with the non-binding contrast X63 immunoprecipitation of MAb CB-IC3/1 (anti-ICAM-3)¹²⁵The cell lysate of the anti-mark of I; (B) with (+) or need not (-) N-dextranase process, and with the non-binding MAb of contrast X63, MAb W6/32 (anti-HLA-A, B, C), MAb RR1/1 (anti-ICAM-1), MAb CBR-IC2/1 (anti-ICAM-2) or MAb CB-IC3/1 (anti-ICAM-3) immunoprecipitation¹²⁵The SKW3 cell lysate of I mark.

Fig. 4 shows the Western engram analysis to the ICAM-3 of the separation of originating from difference. From lymphocyte and neutrophil, separate ICAM-3 by method described in the embodiment. The ICAM-3 that has separated through Polyacrylamide Gel Electrophoresis also uses different antibody to carry out the Western engram analysis.

Fig. 5 shows that PMA is on the impact of SKM3 in conjunction with ICAM-1 and ICAM-3. By the ability of former described method test SKW3 cell in conjunction with ICAM-1 and the ICAM-3 of purifying, and PMA stimulates the impact on this combination.

Fig. 6 shows the SKW3 cell adherence situation that antibody blocking and PMA stimulate. The ability that the SKW3 cell that pressing the various antibody blocking PMA of former described method test stimulates is combined with ICAM-1 or the ICAM-3 of purifying.

Fig. 7 show COS cell transfection body and purifying ICAM in conjunction with situation. By former described method LFA-1 expression vector rotaring redyeing COS cell. Then detect the ability that transfectional cell is combined with immobilization ICAM-1 and ICAM-3 when adding or not adding anti-LFA-1 antibody (TS1/22).

Fig. 8 shows that the SKW3 of PMA stimulation is in conjunction with the temperature dependency of ICAM. Under 4,16,22 and 37 ℃ of conditions, detected the ability that the SKW3 cell of PMA stimulation is combined with ICAM-1 and the ICAM-3 of purifying by former described method.

The impact that the T cell that Fig. 9 displays temperature stimulates PMA is combined with ICAM-3. Under 17,22 and 37 ℃, detect the T cells of PMA stimulation in conjunction with the ability of the ICAM-3 of purifying.

Figure 10 shows the T Cell Differentiation that antibody blocking PHA stimulates. Detected the ability of the T Cell Differentiation of various antibody blocking PHA stimulation by former described method.

Figure 11 shows that anti-ICAM antibody is on the impact of the lymphocyte reaction of mixing. Press the ability of the lymphocyte reaction of the various antibody blockings mixing of former described method test.

Figure 12 shows the gas chromatography result of ICAM-3 fragments of peptides. Purifying also uses Lys-C to digest ICAM-3. Analyzed fragments of peptides by former described method through high performance liquid chroma-tography.

Figure 13 shows clone 11.2 cleavage map. Wherein pointed out the position in NK10 and NK 17 peptides, the various dna sequence dnas that so obtain and cross-film district.

Figure 14 contrasts to NK10 protein and H-ICAM-1's sequence. Use the GAP program to identify the sequence homology of NK-10 protein and H-ICAM-1's protein.

Figure 15 has compared the sequence of RM13 initiation and H-ICAM-1's sequence. Use the GAP program to identify the sequence of RM13 initiation and H-ICAM-1's sequence homology.

Figure 16 has compared the sequence of RM13 initiation and the sequence of people ICAM-2. Use the GAP program to identify the sequence of RM13 initiation and the sequence homology of people ICAM-2.

Figure 17 has compared the sequence of T7 initiation and H-ICAM-1's sequence. Use the GAP program to identify the sequence of T7 initiation and H-ICAM-1's sequence homology.

Figure 18 has compared the sequence of T7 initiation and the sequence of people ICAM-2. Use the GAP program to identify the sequence of T7 initiation and the sequence homology of people ICAM-2.

Figure 19 shows the partial sequence of ICAM-3. Use standard method to detect clone 11.2 dna sequence dna.

A. the sequence that obtains from 5 of ICAM-3 ' end. These sequences cause with the T7 primer and obtain.

B. the sequence that in the ICAM-3 gene, obtains. These sequences cause clone 11.2 with NK-10 probes and obtain.

C. the sequence that obtains from 3 of ICAM-3 ' end. These sequences cause clone 11.2 with reverse RM13 primer and obtain.

The present invention is based on and found in the past still unidentified part of being combined with LFA-1. Such as those molecules of having participated in the molecule adhesion process and being called as the CD-18 family of " adhesion molecule ".

I.LFA-1

In immunity and inflammatory process, the interaction (Springer, T.A.et al.Ann.Rev.Immunol.5:223-252 (1987)) of the various kinds of cell such as leukocyte adhesion molecule LFA-1 mediated leucocytes, monocyte, natural killer cell and granulocyte and other cells.

LFA-1 is the acceptor of ICAM-1, ICAM-2 and the ICAM-3 (as disclosed herein) that recently identifies. These surface moleculars are expressed and be induced to produce (Marlin, S.D.et al., Cell 51:813-819 (1987) at its hetero-organization in inflammatory processes at some tissue; Dustin, M.L.et al., J.Immunol.137:245-254 (1986); Dustin, M.L.et al., Immunol.Today, 9:213-215 (1988); U.s. patent application serial number 07/019,440 (February 7 1987 applying date) and u.s. patent application serial number 07/250,446 (September 28 1988 applying date), these two parts of patent documentations are all classified this paper list of references as).

LFA-1 (the springer that in the interaction of antigentic specificity and antigen dependent/non-dependent T cellular toxicity cell, t helper cell, granulocyte and monocyte and other types cell, plays a role, T.A.et al., Ann.Rev.Immunol.5:223-252 (1987); Kishimoto, T.K.et al., Adv.Immunol. (1988, wait to publish)).

II.ICAM-1

ICAM-1 be present on the different cells molecular weight can be by 76 to the 114KD strand glycoprotein that do not wait, and be member (Dustin, M.L.et al., the Immunol.Today 9:213-215 (1988) that the Ig superfamily in 5 class C zones is arranged; Staunton, D.E.et al., Cell 52:925-933 (1988); Simmons, D.et al., Nature 331:624-62) (1988)). ICAM-1 can be included at an easy rate IFN-g, TNF and IL-1 induces at interior cytokine, produces (Dustin, M.L.et al., Immunol.Toady 9:213-215 (1988)) at the number of different types cell. But induce the LFA-1 dependence of ICAM-1 mediated lymphocytes of generation to adhere to (Dustin, M.L.et al., J.Immunol.137:245-254 (1986) endothelial cell, epithelial cell and fibroblast; Dustin, M.L.et al., J.Cell. Biol.107:321-331 (1988); Dustin, M.L.et al., J.Exp.Med. 167:1323-1340 (1988)). Adhesive attraction (Dustin, M.L.et al., J.Immunol.137:245-254 (1986) can blocked with LFA-1 MAb primed lymphocyte or after with other cells of ICAM-1 MAb preliminary treatment; Dustin, M.L.et al., J.Cell Biol.107:321-331 (1988); Dustin, M.L.et al., J.Exp.Med.167:1323-1340 (1988)). In artificial membrane or plate prove that with the result that the ICAM-1 of purifying identifies LFA-1 and ICAM-1 be acceptor (Marlin, S.D.et al., Cell 51:813-819 (1987) each other; Makgoba, M.W. et al., Nature 331:86-88 (1988)). For the sake of clarity, they are called " acceptor " and " part " herein. In the u.s. patent application serial number 07/045,963,07/115,798,07/155,943,07/189,815 or 07/250,446 (all these Patent Application Publications are all classified this paper list of references in full as) ICAM-1 has been done further description.

III.ICAM-2

Other LFA-1 parts (Rothlein, R.et al., the J.Immunol.137:1270-1274 (1986) that are different from ICAM-1 have been inferred; Makgoba, M.W.et al., Eur.J.Immunol.18:L637-640 (1988); Dustin, M.L.et al., J.Cell Biol.107:321-331 (1988)). The second LFA-1 part of identifying is named is " ICAM-2 ".

ICAM-2 distributes and lacks cytokine at cell and is different from ICAM-1 aspect inducing two. ICAM-1 is the integration memebrane protein with 2 class Ig zones, and ICAM-2 then has 5 class Ig zones, and (Cell 52:925-933 (1988) for Staunton, D.E.et al.; Simmons, D.et al., Nature 331:624-627 (1988)). The more important thing is that ICAM-2 is more closer than ICAM-1 or ICAM-2 and other members' of Ig superfamily relation with the relation of two N stub areas of ICAM-1, has proved that the subfamily of class Ig part combines same integration factor family receptors. In the u.s. patent application serial number 07/454,294 ICAM-2 has been done further description (this paper list of references is classified in this patent application as).

IV.ICAM-3

The present invention relates to find be called the third LFA-1 part of " ICAM-3 ".

The MAb that develops anti-ICAM-2 can be analyzed several LFA-1 dependence, ICAM-1 dependent/non-dependent phenomenons of observing in the past, and there is the third part of LFA-1 in prompting. Unite and use ICAM-1 and ICAM-2 MAb can block the combination of the several types cells such as epithelial cell and endothelial cell and the IFA-1 of purifying fully, comprise that many lymphoid cells of t cell lymphoma clone SKW3 are fastened ICAM-1, the ICAM-2 dependent/non-dependent adhesion approach (Fig. 1) that then has for LFA-1.

In order to illustrate this adhesion approach, produced the anti-SKW3 MAb consistent with anti-ICAM-1 and anti-ICAM-2 MAb, and suppressed this clone according to it and screen it with the ability that the LFA-1 of purifying combines. A kind of MAb that selects, namely CBR-IC3/1 can suppress this new adhesion approach (Fig. 1) fully. Unite and use the anti-ICAM-1 of blocking-up property and anti-ICAM-2 MAb can only slightly suppress SKW3 to the adhesion of the LFA-1 of purifying. When the anti-ICAM-3 MAb of independent adding (CB-IC3/1), can suppress significantly to adhere to, and when share with the anti-ICAM-2 MAb of blocking-up property, can reach fully inhibition. Therefore, SKW3 is mediated by ICAM-3 the sticking to a great extent of LFA-1 of purifying, and a part is mediated by ICAM-2. As suppress the Curves proof according to the difference of MAb, in the adhesion to LFA-1, four clones are being utilized this three kinds of ICAM respectively in varying degrees. The adhesion of B lymph matricyte system JY mainly is by ICAM-1 approach, and ICAM-2 and ICAM-3 also have certain contribution. Another B lymph matricyte system SLA has then utilized ICAM-1 and ICAM-3, because can not suppress to adhere to ICAM-1 or ICAM-3 MAb separately, could suppress fully when only share two kinds of MAb. Thymoma has used ICAM-2 and ICAM-3 approach when being the Jurkat adhesion, and ICAM-1 also has less contribution. Also studied static T lymphocyte and with the lymphocytic adhesive attraction of T (Figure 1B) of lectins (PHA) activation. The static lymphocyte that studies show that in the past can be combined forcefully with the LFA-1 of purifying (Dustin et al., Nature 341:619-624 (1989)), and finds that this being combined in depends on ICAM-3 to a great extent. After with the PHA activation, induced ICAM-1 to express and the main ICAM-1 of passing through, and partly by the adhesion of ICAM-3 generation to LFA-1. In static and the T cell that activates, although the ICAM-2 composition that adheres to is covered because of the existence of ICAM-1 and ICAM-3, but still can detect. Because for every kind of cell, need at least the antibody of anti-two kinds of ICAM for reaching basic inhibition, so can the relevant composition of considerable residue in using ICAM. Noticeable exception is that static lymphocyte is mediated by ICAM-3 sticking to a great extent of LFA-1. In all cases, share the combination of three kinds of anti-ICAM MAb eliminatings and LFA-1, and few with the level error that in the presence of anti-LFA-1 MAb, reaches.

Can compare three kinds of ICAM in conjunction with the contribution (detecting as top) of LFA-1 and their the cell surface expression situation that records with the immunofluorescence flow cytometry, with the relative compatibility (Fig. 2) of definite three kinds of ICAM to LFA-1. The relatively surface expression of ICAM and they contribution that LFA-1 is adhered to discloses ICAM-1 LFA-1 is had larger compatibility, and ICAM-2 and ICAM-3 then have similar but lower compatibility. For example, the Jurkat cell can be expressed ICAM-and ICAM-3 with similar level, and separately to making contributions with the combination of LFA-1. When ICAM-2 expresses expression greater than ICAM-3 (as on the JY cell), the ICAM-2 approach that LFA-1 adheres to namely surpasses ICAM-3. On the contrary, the ICAM-3 that SLA and SKW3 express manys 2-5 doubly than ICAM-2, and this point is fully understandable, because the ICAM-3 approach that adheres to is better than the ICAM-2 approach. ICAM-3 can well be expressed at static T lymphocyte, and does not have ICAM-1, and the adhesion of LFA-1 is mainly mediated by ICAM-3. When giving full expression to ICAM-1 (in the situation such as the T cell that activates at SLA, JY and PHA), just ICAM-1 has become the main path that adheres to.

The distribution of ICAM-3 is different from ICAM-1 and ICAM-2 in several ways. Different from ICAM-1 and ICAM-2, ICAM-3 does not express (data is not shown) at endothelial cell static or that activate. This point conforms to following discovery: namely to be combined with the LFA-1 dependence of the endothelial cell of static and irriate be a kind of ICAM-1 and ICAM-2 dependence phenomenon to cell. ICAM-3 is only limited to hematopoietic cell lineage, and except minority exception, it can be fastened highly at lymphoid cell and monocyte and be expressed. But in all cases, the expression of ICAM-3 on clone is all consistent with the LFA-1 dependence, ICAM-1, the ICAM-2 dependent/non-dependent approach that adhere to. Only do not show the expression of ICAM-3 by ICAM-1 and ICAM-2 in conjunction with the clone of LFA-1, show that the clone (JY, U937, SupT) that produces faint combination by the 3rd adhesion approach then has corresponding low ICAM-3 surface expression (data is not shown).

With regard to the expression of ICAM-3 on leucocyte, it obviously is different from ICAM-1 and ICAM-2 (Fig. 2 B). ICAM-3 can express static lymphocyte, monocyte and neutrophil(e) cell with high level, but the expression of ICAM-1 and ICAM-2 then wants how weakly or do not express. By relatively, can faintly express ICAM-1 and ICAM-2 on the monocyte as can be known, and only have ICAM-2 to be present on the static lymphocyte. ICAM-1 and ICAM-2 all do not express the neutrophil(e) cell. After with the PHA activated lymphocyte, ICAM-3 expresses has increased 2-3 doubly, and ICAM-1 expresses and then induced significantly (Dustin et al., J. Immunol.137:245-254 (1986)) (Fig. 2 B).

Derive from various¹²⁵The ICAM-3 immunoprecipitate of the clone of I mark presents the clear band of a molecular weight about 124,00000 under reducing condition, only increased a little mobility (Fig. 3 A) in non-reduced condition simultaneously. Produce the band (Mr 87,000) of the ICAM-3 of lower molecular weight after processing with the N-dextranase, this show ICAM-3 the same with ICAM-1 and ICAM-2 be the protein (Fig. 3 B) of high glycosylation. The adhesion molecule of describing before the biochemical characteristic of ICAM-3, expression pattern and adhesion character all are different from, comprise people acceptor LAM-1 (the Tedder et al. that " goes back to the nest ", Immunol.144:532-540 (1990)), derivable endothelial cell adhesion molecule VACAM-1 (Rice et al., J.Exp.Med.171:1369-1374 (1990); Carlos et al., Blood (1990, wait to publish); Osborn et al., and matrix acceptor (Hemler, the M.E. of VLA family Cell 59:1203-1211 (1989)),, Ann.Rev.Immunol. 8:365-400 (1990)), and the non-MAb (Gilks, W.R., the et al. that see the similar cell distribution of tool in the Sibai cell database, Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England (1990)).

Exist this true prompting of three kinds of LFA-1 parts to specialization of the different aspect of LFA-1 dependence leucocyte interaction. ICAM-1 expresses at endothelial cell and many type epithelial cells, and under inflammation and immune condition, induced by force, to regulate celluar localization and to be conducive to specific antigen identification (Wawryk et al., Immunol.Rev. 108:135-161 (1989)). Because ICAM-2 is the main LFA-1 part on the static endothelial cell, so should the adhesion approach for the lymphocyte that carries LFA-1 by organizing endothelial cell normally to recycle significant (Hamann et al., J. Immunol.140:693-699 (1988); Mackay et al., J.Exp.Med.171:810-817 (1990); Pals et al., J.Immunol.140:1851-1853 (1988); Nunoi et al., Hum.Path.19:753-759 (1988)). The function of ICAM-3 on the neutrophil(e) cell is still unclear at present. As if although there is LFA-1, neutrophil's homotypic aggregation mainly is (Anderson et al., the J.Immunol. 137:15-27 (1986) that relies on Mac-1; Patarroyo et al., Scand.J.Immunol.22:619-631 (1985)). Static T lymphocyte mainly adheres to LFA-1 by ICAM-3, and ICAM-3 can express better than other LFA-1 part on monocyte and static lymphocyte, and these are found and the fact shows that it has important function in starting immune response. The adhesion of T cell and antigen presentation cell needs to have LFA-1:ICAM interaction (Dransfield et al., Imm.Rev.114:29-44 (1990) really; Makgoba et al., Immunol.Today 10:417-422 (1989)), and on static T cell more may be ICAM-3 in action because can not express well ICAM-1 and ICAM-2 on the type cell. Really, in allos and AMLR, prompting is LFA-1 part rather than ICAM-1 in action (Bagasco et al., Cell Immunol.128:362-369 (1990)). In addition, infer that it should be very important that the antigentic specificity of ICAM-3 between T and bone-marrow-derived lymphocyte interacts, because wherein a kind ofly not yet be activated this moment. ICAM-3 can depend on LFA-1 but not rely on play an important role in the dissolving of T cell to some target cell of ICAM-1 (Makgoba et al., Eur.J.Immunol.18:637-640, (1988)).

There is multiple ICAM, carries out clinical treatment for the MAb that uses anti-ICAM or ICAM analog and have important association. ICAM-1 MAb prolongs kidney (Cosimi et al. in vivo, J.Immunol.144:4604-4612 (1990)) and be effective in the heart (Flavin et al., Transplant.Proc.23:533-534 (the 1991)) homograft. Because can suppressing the LFA-1 dependence of different subgroup cell types, ICAM-3 MAb interacts, so can be used for suppressing in vivo the immune response of specific and many intersections.

The cDNA clone of V.ICAM-3

Useful several different methods is cloned the ICAM-3 gene. Wherein a kind of method is to analyze the shuttle vector library of cDNA insert (be derived from ICAM-3 and express cell), with existing of the insert of determining to contain the ICAM-3 gene. Then the available support transfectional cell detects the expression of ICAM-3, to carry out this analysis.

Preferably use improving one's methods of Aruffo and Seed (Seed, B.et al., Proc.Natl.Acad. Sci.USA 84:3365-3369 (1987)) to identify ICAM-3 cDNA, to identify the part of adhesion molecule. In the method, preparation cDNA library from the cell (such as SLA, Jurkat or SKW3 lymph matricyte system) of expressing ICAM-3. Be more preferably from the T cell and prepare cDNA library. Do not express under normal circumstances the cell (such as Cos or Hela cell) of ICAM-3 with this library transfection. The cell of transfection is introduced in advance in the plate with LFA-1 or anti-ICAM-3 antibody sandwich. Contain the ICAM-3 coded sequence, and will adhere on the lip-deep LFA-1 of plate or the anti-ICAM-3 antibody at the transfected cell of its this part of cell surface expression. Wash non-adherent cell off, then from plate, remove adherent cell and cultivate it. Then the recombinant ICAM-3 that removes in these its sequences of cell inner expression also carries out sequence analysis.

As with the coated plate of LFA-1, can in plate, add anti-ICAM-1 and anti-ICAM-2 specific antibody, express the adhesion of cell to prevent ICAM-1 or ICAM-2. So available antibodies such as RR1/1 (anti-ICAM-1 MAb) and CBR-1C2/2 (anti-ICAM-2 MAb) suppress ICAM-1⁺Or ICAM-2⁺Transformant adheres on the coated plate of LFA-1. The combination of the plate that the cell of ICAM-3 transfection and LFA-1 are coated can be suppressed by EDTA and anti-LFA-1 MAb, but can not be suppressed by anti-IACA-1 or anti-ICAM-2 MAb. Therefore ICAM-1 or ICAM-2 express cell and can not adhere on the plate, thus great majority can be washed off with every other non-adherent cell, thereby enrichment express the cell of ICAM-3.

Other methods selected that obtain the gene order of coding ICAM-3 are as known in the art, and those skilled in the art can select wherein, and a kind of method obtains required gene.

A kind of method that obtains the gene order of coding ICAM-3 is to use oligonucleotide probe screening cDNA or genomic library. In the method, can use purification process purifying ICAM-3 protein as known in the art, and use method as known in the art to measure its terminal amino acid sequence. In addition, can prepare the gene map of ICAM-3, and measure the amino acid sequence of one of inherent fragment.

In case measured partial amino-acid series, can prepare oligonucleotide probe based on the preferred codon that organism is showed, perhaps based on all possible codon combination preparation degeneracy probe. Then screen genome or the cDNA library of its sequence and Probe Hybridization with this probe.

Also can use in addition genome obtain the encoding cDNA clone (Watson of ICAM-3, J.D., In:Molecular Biology of the Gene, 3rd Ed., W.A.Benjamin, Inc., Menlo Park, CA (1977), pp.356-357), with the poly thuja acid probe of the fragments of peptides amino acid sequence of determine to encode ICAM-3 protein or ICAM-3 protein. Then with this probe detect in the member of coding ICAM-3 protein cDNA library (from separating the mRNA preparation of expressing cell from ICAM-3) or the detection genome dna library can with the genome sequence of this oligonucleotide hybridization.

Use successfully clones coding people aldehyde dehydrogenase (Hsu of above-mentioned technology or above-mentioned similar technique, L.C.et al., Proc.Natl.Acad.Sci.USA 82:3771-3775 (1985)), Fibronectin (Suzuki, S.et al., Eur.Mol.Biol.Organ. J.4:2519-2524 (1985)), people's ERs (Walter, P.et al., Natl.Acad.Sci.USA 82:7889-7893 (1985)) and (Pennica of tissue-type plasminogen activator Proc., D.et al., Nature 301:214-221 (1983)) gene.

In the another kind of method of clone ICAM-3 gene, can derive from through the clone genomic DNA of the cell of expressing ICAM-3, be more preferably cDNA and prepare expression library. Then screen the member that can express the protein of being combined with anti-ICAM-3 antibody in the library.

The clone's that makes by above-mentioned either method ICAM-3 gene can be operably connected on the expression vector, and import in bacterium or the eukaryotic to produce ICAM-3 protein. Gene manipulation techniques can be referring to Maniatis, the people's such as T. above-mentioned document, and these technology all are as known in the art.

VI. reagent of the present invention: ICAM-3 exhales basic functional derivatives, activator and antagonist

The invention further relates to ICAM-3, its " functional derivatives ", its " activator " and " antagonist ".

The functional derivatives of A.ICAM-3

" functional derivatives " and the ICAM of ICAM has the compound of basically similar BA (function or structure). Term " functional derivatives " comprises " fragment ", " variant " and " chemical derivative " of parental generation ICAM-3 molecule.

" fragment " of ICAM-3 refers to the polypeptide subgroup of molecule. The fragment of ICAM-3 preferably has the ICAM-3 activity and is (namely not with membrane-bound) of solubility. Soluble fragments better is to replace the hydrophobic residue generation by the cross-film district of deletion parental generation molecule or with hydrophilic amino acid residue. The method of identifying these residues is as known in the art.

" variant " of ICAM-3 refers on the 26S Proteasome Structure and Function basically similar to whole molecule or its fragment molecule.

If two molecules have basically similar structure or BA, can say that then a molecule " basically similar " is in another molecule. Therefore, with regard to term used herein, even a molecule has the structure that does not have in another molecule, or this sequence has incomplete same amino acid residue, as long as two molecules have similar activity, also is considered to variant.

With regard to term used herein, when a molecule contained the additional chemical part of the normal part that is not natural molecule, then to be said to be " chemical derivative " of another molecule to this molecule. Said part may be improved the dissolubility, adsorptivity of molecule and biology half life etc. This part also may be fallen low molecular toxicity or elimination or be weakened harmful pair of effect of molecule. The molecular moiety that can mediate this effect is disclosed among the Remington ' s Pharmaceutical Sciences (1980).

" toxin is derived " molecular composition special " chemical derivative " of one class. " toxin is derived " molecule is the molecule (such as ICAM-3 or anti-ICAM-3 antibody) that is covalently bound on the toxin moiety. The method of these parts of coupling is as known in the art.

Such molecule is connected on the cell, thereby can makes toxin moiety impel cell death closer to cell. Can use any suitable toxin moiety; But better be to use ricin toxin, cholera toxin, diphtheria toxin, radionuclide toxin or film passage to form toxin.

Can use conventional synthetic method to have nearly ICAM-3 functional derivatives of 100 residues in external preparation. In case of necessity, available known method is modified such fragment, comprises making target amino acid residue purifying or rough protein and the organic chemistry dressing agent reaction that can react with side chain or the terminal residue of selection. Can use the gained covalence derivative to identify that those are for the very important residue of BA.

Can produce in many ways and separate the fragment of ICAM-3. Can use bifunctional reagent that ICAM-3 is linked on the non aqueous carrier matrix. In addition, can use the non-water-soluble matrix of carbohydrate isoreactivity and the United States Patent (USP) 3,969,287,3,691 of cyanogen bromide activation, 016,4,195,128,4,247,642,4,229,537 and 4,330, the reactive matrix described in 440 fixes protein.

The functional derivatives that also can prepare by the DNA of sudden change coding ICAM-3 the ICAM-3 that has changed amino acid sequence. For example, such functional derivatives comprises the forms such as disappearance, insertion or replacement of the interior residue of amino acid sequence of ICAM-3. Any combining form that can utilize disappearance, insert and replace produces last construct, as long as this structure people has required activity. Obviously, the sudden change that will cause in the DNA of encoding function derivative necessarily can not make sequence be in outside the reading code, and preferably can not cause the complementary region that will produce secondary mRNA structure (referring to EP Patent Application Publication numbers 75,444)

Can carry out direct mutagenesis by the DNA to coding ICAM-3, to produce the DNA of encoding function derivative, then in the recombinant cell culture, express this DNA, thereby prepare functional derivatives at gene level.

Although can pre-determine the site of introducing sudden change in amino acid sequence, sudden change itself does not need to pre-determine. For example, to reach optimum efficiency in order making to the sudden change on the anchor point, can to carry out random mutagenesis in target codon place or target region such as mutagenesis is scanned in introducing, producing the derivatives that can express thereafter in a large number, and screening has required the drawing together property of best of breed. It is known that predetermined site in known dna sequence causes the technology of sudden change, and for example specificity mutagenesis method is decided in the position.

The preparation of the functional ICAM-3 derivative of the present invention, preferably by to coding ICAM-3 or early the DNA of the functional derivatives of the ICAM-3 of preparation carry out direct mutagenesis and finish. Side-directed mutagenesis is as known in the art, as referring to Maniatis, and T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, NY (1982), the document is classified this paper list of references as. Direct mutagenesis allows us to produce the functional derivatives of ICAM-3 by the specific oligonucleotide sequence of the dna sequence dna of the required sudden change of encoding.

Can use the site-specific mutagenesis method to produce amino acid deletions, insertion or replacement. Sequential amino acid deletion generally is about 1-30 residue, better is 1-10 residue, and generally is continuous. Most preferred disappearance is those disappearances that can produce solubility ICAM-3. Preferably by the cross-film zone of disappearance ICAM-3 or the ICAM-3 that the hydrophobic residue in the ICAM-3 produces soluble form.

Amino acid insert be included in insert in the ICAM-3 coded sequence single or multiple amino acid residues and with basically not more than limit for length's degree peptide carry out end fusion. The sequence scope of inserting (namely inserting) in the sequence in complete ICAM-3 molecular sequences is generally 1-10 residue, is more preferably 1-5 residue. A terminal example that inserts comprises with single allos or with the sequence that comes from host cell and being fused on the N end of molecule, is beneficial to from restructuring host endocrine derivative.

The 3rd group of functional derivatives is those derivatives that removed the one or more amino acid residues in the ICAM-3 molecule and inserted different residues in this position. Adjust more accurately the feature of ICAM-3 molecule such as expectation, but according to the form below replaces preferably.

Table 1

The replacement that original residue is given an example

Ala                                           gly；ser

Arg                                           lys

Asn                                           gln；his

Asp                                           glu

Cys                                           ser

Glu                                           asn

Glu                                           asp

Gly                                           ala；pro

His                                           asn；gln

Ile                                           leu；val

Leu                                           ile；val

Lys                                           arg；gln；glu

Met                                           leu；tyr；ile

Phe                                           met；leu；tyr

Ser                                           thr

Thr                                           ser

Trp                                           tyr

Tyr                                           trp；phe

Val                                           ile；leu

Can select has the still less replacement of conservative than amino acid shown in the table 1, namely select those for keeping (a) to replace polypeptide backbone structure in the zone, such as lamella or helical conformation, (b) charging property or molecule hydrophobicity on the target site, or (c) the side chain size plays obviously the not residue of same-action, carries out material alterations with function or immunology character to molecule. In general, less conservative replacement be those wherein (a) glycine and/or proline by another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, or disappearance or be inserted into; (b) replace hydrophobic residue with hydrophilic residue; (c) replace (or being substituted) any other residue with cysteine; (d) replace the residue that (or being substituted) has negative electrical charge with the residue with positive charge side chain; Or (e) to replace (or being substituted) with the replacement of the residue of such side chain with the residue of bulky side chain.

Most preferably can affect the replacement of the solubility of ICAM-3, and preferably hydrophobic amino acid produces by replacing with hydrophilic amino acid.

For improving the compatibility of ICAM-3, it is same to design sudden change according to the guiding theory that introducing is present in the locational amino acid residue of homology among ICAM-1 or the ICAM-2, can prepare this class and listen the mutant ICAM-3 molecule that lacks the CHO of N connection with the source position at IACM-1 or ICAM-2.

Before carrying out these work, be difficult to predict any specific replacement, disappearance or insert the accurate impact that will produce the BA of ICAM-3. But it will be understood by those skilled in the art that, can estimate its effect by conventional screening technique. For example, typically can carry out by the DNA to the natural ICAM-3 molecule of encoding joint scans direct mutagenesis and makes derivative. Then in the restructuring host, express this derivative, and for example utilize immune-affinity chromatography from cell culture purifying it.

Then with the activity of suitable screening technique according to the derivative of expection Feature Selection cell lysate or purifying. For example, the change of the immunological characteristic of available competitive immunization detection method detection functionality derivative is such as the change to the compatibility of known antibody. Available method known to a person of ordinary skill in the art changes the character of this protein, such as redox or heat endurance, biology half life, hydrophobicity, to the sensitiveness of proteolytic degradation effect, or with the carrier polymerization or aggregate into polymeric tendency etc.

The activator of B.ICAM-3 and antagonist

" activator " of ICAM-3 refers to improve or to increase the compound that ICAM-3 finishes the ability of its any biological function, for example can improve the preparation of the ability that ICAM-3 is combined with cell receptor.

" antagonist " of ICAM-3 refers to and can reduce or stop ICAM-3 to finish the compound of the ability of its any biological function. The example of such antagonist comprises ICA M-1 and ICAM-2, the functional derivatives of ICAM-1 and ICAM-2, anti-ICAM-3 antibody, anti-LFA-1 antibody etc.

U.s. patent application serial number 07/045,963,07/115,798,07/115,943,07/189,815 or 07/250, cell aggregation test described in 446 (this paper list of references is all classified in these patent applications in full as) can detect the LFA1 dependence to be assembled, and therefore can be used for identifying the preparation that affects ICAM-3/ LFA-3 aggregation extent. Therefore, these detection methods can be used for identifying activator and the antagonist of ICAM-3. Antagonist can play a role by the ability of recovering LFA-1 or ICAM-3 mediation gatherings. In addition, available above-mentioned method detects NIg (i.e. chemistry) preparation, to determine that they are whether as activator or the antagonist of the ICAM-3/ LFA-1 that mediates gathering. This cell aggregation detection method generally is to use the periphery blood T cell that stimulates with PMA to finish. Therefore, can detect according to the combination of PBC and LFA-1 cell aggregation of ICAM-3 mediation.

C. anti-ICAM-3 antibody

The preferred immunoglobulin (Ig) antagonist of the present invention is anti-ICAM-3 antibody disclosed herein such as CBR-1C3/1. Can make ins all sorts of ways makes other applicable anti-ICAM-3 antibody.

The antibody of anti-ICAM-3 (or its functional deriv) can be polyclone or monoclonal. In addition, can by methods known in the art or " TCT/US91/02942 and PCT/US91/02946 (the U.S. accept the office the applying date be on April 22nd, 1991) in disclosed method make antibody of the present invention that the characteristic of people's antibody must be arranged.

ICAM-3 or its fragments of peptides can be introduced and make anti-ICAM-3 antibody in the suitable animal body. Immunized animal will produce polyclonal antibody in to its course of reaction. Use the fragments of peptides of ICAM-3 only can make and the regiospecificity antibody that reacts for epitope contained in the fragments of peptides of immune animal.

In addition, can use the ICAM-3 that on lymphocytic cell surface, naturally expresses to make anti-ICAM-3 antibody. Such cell is introduced (as by intraperitoneal injection) in the suitable animal body, will produce the antibody that to be combined with the member of ICAM-3 or CD-18 molecule family. Can gather in the crops in case of necessity these animals serum and as can with the source of clonal antibody more than these molecules are combined.

Moreover, can be by Selden, the people's (Scienee 236:714-718 (1987)) such as R.F. (european patent application publication number 289,034) or Selden R.F. method produces anti-ICAM-3 antibody. Specifically, available can the expressed ICAM-3 molecule or the cell of the suitable animal of carrier transfection of its fragment. In the transfected cell of animal, produce ICAM-3 and will cause in animal body immune response, and then produce anti-ICAM-3 antibody by this animal.

Yet, better be in the animal body of as stated above immunity, to remove splenocyte, can be in conjunction with the monoclonal antibody of ICAM-3 to produce.

Available several different methods is identified the hybridoma that can secrete the antibody that can be combined with ICAM-3, to screen the hybridoma that obtains as stated above. In a preferred screening technique, can suppress the ability that ICAM-3 expression, ICAM-1 and ICAM-2 non-expressing cell assemble according to them and identify these molecules. Then further screening can suppress the antibody of this aggtegation, to determine whether they can be by being combined to suppress this aggtegation with the member of ICAM-3 or CD-18 molecule family. In screening, can use any method that ICAM-3 and CD-18 molecule family can be distinguished. Therefore, for example available immuno-precipitation and polyacrylamide gel electrophoresis analysis by the antigen of antibody combination. Might according to antibody with express LFA-1 but the ability of the cell combination of non-Table I CAM-3 (or opposite) is distinguished the antibody of being combined with CD-18 molecule family member and the antibody of being combined with ICAM-3. The method that can commonly use by those of ordinary skills detects antibody with expression LFA-1 but does not express the ability that the cell of ICAM-3 combines. These methods comprise immune detection method (particularly using immunofluorescence), cell agglutination method, filter membrane combined techniques and antibody precipitation method etc.

In this preferred method, according to its cell in conjunction with expression ICAM-3, but do not select antibody in conjunction with the ability of not expressing the cell of ICAM-3.

Except the activator and antagonist of above-mentioned ICAM-3, can be used according to the invention as the antiantibody of anti-ICAM-3 antibody, can be in conjunction with other preparations treatment inflammation, HIV infection, asthma etc. such as the acceptor molecule of ICAM-3 or its fragments.

The significant anti-genotype antibody of the same race of the present invention should be able to be competed with ICAM-3 (or repulsion) combination. For example, can induce the antiantibody of anti-ICAM-3 antibody, then screening has the antibody in conjunction with the ability of the binding partner of natural ICAM-3, to obtain such antibody.

Because the molecule of CD-18 family can be combined with ICAM-3, so use this molecule (as have the heterodimer of α and β subunit, or the molecule that is only formed by α or β subunit or the molecule fragment with one or both subunit) to be combined with cell competition to be present in the ICAM-3 on the cell.

Available several different methods is identified and titration anti-aggtegation antibody of the present invention. For example, can detect discriminatively combination and express the cell (such as the T cell) of ICAM-3, and can not be in conjunction with the antibody of the cell of not expressing ICAM-3. U.s. patent application serial number 07/ 045 has been described the method (this paper list of references is all classified in all these patent applications as) that is suitable for detecting cell aggregation in 963,07/115,798,07/155,993,07/189,815 or 07/250,446. In addition, the detectable antibody ability of being combined with the fragments of peptides of ICAM-3 or ICAM-3. Those of ordinary skills can understand at an easy rate, can improve above-mentioned detection method, or finish it by different order, so that multiple feasible screening technique to be provided, and wherein each method all can be differentiated the antibody that can be combined with ICAM-3 and distinguish with the antibody that can be combined with CD-18 molecule family member.

D. the preparation of preparation of the present invention

Can make preparation of the present invention by several different methods: natural method (as induce animal, plant, fungi, bacterium etc. to produce the NIg antagonist of ICAM-3, or induce animal to produce the polyclonal antibody that can be combined with ICAM-3); Synthetic method (for example functional derivatives of synthetic ICAM-3, ICAM-3 or the protein antagonist (immunoglobulin (Ig) or NIg) of ICAM-3); Hybridoma technology (as producing the monoclonal antibody that to be combined with ICAM-3); Or restructuring technology (as using recombinant plasmid or viral vectors generation preparation of the present invention in different host (such as the mammalian cell of yeast, bacterium, fungi, cultivation etc.)). Select which kind of method whether will depend on the factors such as convenient and productive rate is big or small. But not necessarily only produce specific anti-inflammatory preparation with above-mentioned a kind of method or technology; Can unite above-mentioned method and the technology used for obtaining specific preparation.

The application of VII, ICAM-3 and functional derivatives thereof, activator and antagonist

It is active to use preparation of the present invention to regulate by the various biological of ICAM-3 mediation.

A. suppress inflammation

A method of the present invention is based on the particularly interactional ability of acceptor of LFA-1 of ICAM-3 and its functional derivatives and CD 18 family molecules. By ICAM 3 and the interactional ability of CD-18 glycoprotein family member, it can be used to control (namely stop or weaken) inflammation.

Term " inflammation " refers to the reaction of specificity system of defense, and the reaction of nonspecific defense system.

Term used herein " specificity system of defense " refers to the immune composition that the existence to specific antigen reacts. If inflammation causes mediation by the specificity system of defense, or be associated with the specificity system of defense, then this inflammation is counted as the result of the reaction of specificity system of defense. The inflammation that produces because of the reaction of specificity system of defense comprises the antigen delayed allergy (as seen in Mantaux test " positive " person) of reaction, oneself immunological disease and the mediation of T cell of rubella virus for example. Other examples of the inflammatory reaction of specificity system of defense comprise that the chronic inflammation disease reaches the rejection to entity transplanted tissue and organ (bribing transplanting such as kidney and bone).

The reaction of this paper said " nonspecific defense system " refers to the reaction by the leucocyte mediation that can not produce immunological memory. This class cell comprises granulocyte and macrophage. As described herein, if inflammation is reaction by the nonspecific defense system to be caused or mediate, or contact with the reacting phase of nonspecific defense system, then namely to say into be the result of non-specific system of defense reaction to this inflammation. At least part of is because the example of the inflammation that the reaction of nonspecific defense system causes comprises the inflammation relevant with following pathological conditions: the reperfusion injury of ARDS (ARDS) or septicemia, wound or posthemorrhagic multiple organ injury's syndrome, cardiac muscle or its hetero-organization, acute glomerulonephritis, adjuvant arthritis, with skin disease, acute purulent meningitis or other inflammatory disease of central nervous system of acute inflammation change such as apoplexy, fire damage, blood are rolled over thoroughly, leucocyte deformity, ulcerative colitis, regional ileitis, NEC, granulocyte are transferred related syndrome and cytokine is induced toxicity.

As mentioned above, being combined in of ICAM-3 molecule and CD-18 molecule family member played key effect in the cell adherence. By adhesion process, lymphocyte can monitor the existence of external antigen in the animal body continuously. Although needing under normal circumstances has these processes, they can cause also that (such as marrow) is repelled in parenchymatous organ's graft rejection (such as kidney), unsubstantiality organ transplant thing, the tissue transplantation thing repels and many oneself immunological diseases. Therefore, the way that can weaken or suppress cell adherence is found in patient or the special expectation of oneself immune patient of carrying out parenchymatous organ's transplanting (particularly kidney transplant), unsubstantiality organ transplant (particularly bone-marrow transplantation), tissue transplantation.

Anti-CD-18 molecule family member's monoclonal antibody can suppress leukocytic many adhesion dependence functions, comprise the combination (Haskard with endothelial cell, D.-et al., J. Immunol.137:2901-2906 (1986)), homotype adheres to (Rothlein, R.et al., J.Exp.Med.163:1132-1149 (1986)), lymphopoiesis (the Davignon that antigen and mitogen are induced, D.et al., Proc.Natl.Acad.Sci.USA. 78:4535-4539 (1981)), antibody generates (Fisher, A.et al., J.Immunol. 136:3198-3203 (1986)), and all leukocytic effector molecules functions, such as cellular toxicity T cell (Krensky, A.M.et al., J.Immunol.132:2180-2182 (1984)), macrophage (Strassman, G.et al., J.Immunol.136:4328-4333 (1986)) and all participate in the cell (Kohl of antibody dependent cellular cellular toxicity reaction, S.et al., J.Immunol.133:2972-2978 (1984)) lytic activity. In all above-mentioned functions, antibody can suppress the ability that leucocyte adheres to suitable cellular matrix, and the latter suppresses again the biological function that is associated with above-mentioned combination conversely. Can participate in the interactional degree of ICAM-3/LFA-1 according to them, suppress these functions with anti-ICAM-3 antibody.

Therefore, can use the monoclonal antibody that to be combined with ICAM-3 as mammiferous anti-inflammatory preparation. This class preparation is that from the obvious different of common antiinflammatory they can optionally suppress adhesive attraction, not have common other pairs effect (such as nephrotoxicity) of conventional dose.

Because ICAM-3 (the particularly ICAM-3 of soluble form) can work with the same manner of anti-CD-18 molecule family member's antibody, so also can be in order to suppress inflammation. In addition, also can use the functional derivatives of ICAM-3 and antagonist to suppress inflammation.

1. the inhibitor of delayed allergy

ICAM-3 partly mediates as forming inflammatory reaction such as the necessary adhesion process of delayed type hypersensitivity, DTH. Therefore, the antibody that can be combined with ICAM-3 weakening or eliminate these the reaction in have therapeutic value.

In addition, because the ICAM-3 interactional antagonist that is ICAM-1/LFA-1, so can use ICAM-3 (particularly its soluble form) or its functional derivatives to suppress delayed type hypersensitivity, DTH.

Can develop these potential treatments by dual mode uses. The first, can there be the patient of delayed type hypersensitivity, DTH to use the composition that contains anti-ICAM-3 monoclonal antibody or solubility ICAM-3 derivative to experiment confirm. For example, use this composition can for the patient of the antigens such as contacted malicious ivy, poison oak. In another embodiment, can share to patient can be in conjunction with monoclonal antibody and the antigenic substance of ICAM-3, to prevent the Secondary cases inflammatory reaction. Therefore, additional antigen in anti-ICAM-3 antibody, the antigen that the temporary transient tolerance of patient will be contacted again.

2. treatment chronic inflammatory disease

Patient LAD in default of LFA-1 can not produce inflammatory reaction, also will suppress inflammatory reaction so can believe the antagonism of the native ligand of LFA-1. The ability of anti-ICAM-3 antibody suppression inflammation is used for their treatments in oneself immunological diseases such as the diabetes for the treatment of chronic inflammation disease and lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some type, Lei Nuoshi syndrome, rheumatoid arthritis and is provided the foundation. Also can use these Antybody therapy psoriasis.

Generally speaking, anti-ICAM-3 antibody of the present invention can be individually dosed, also can with anti-ICAM-1 and/or the antibody combined use of anti-ICAM-2, being used for the treatment of those at present can be by the disease of Steroid treatment treatment.

According to the present invention, can give need the object for the treatment of that the antiinflammatory that suppresses the inflammation q.s is provided, in order to suppress this inflammatory and immune response (namely stop or weaken it). Applicable antiinflammatory comprises: the fragment that can be combined with ICAM-3 of the antibody that can be combined with ICAM-3, said antibody, basically functional derivatives, the NIg antagonist of ICAM-3 or the NIg antagonist of the ICAM-3 beyond the LFA-1 of pure ICAM-3, ICAM-3. Particularly preferably be the antiinflammatory that the soluble functional derivative by ICAM-3 forms. Such anti-inflammatory treatment can comprise using to patient and is attached with the composition that is selected from following preparation: the antibody that can be combined with LFA-1, the NIg antagonist of LFA-1, pure ICAM-1 and/or ICAM-2 or derivatives thereof basically, or anti-ICAM-1 and/or anti-ICAM-2 antibody or its fragment.

The present invention further comprises the method for the inflammatory reaction of above-mentioned inhibition specificity system of defense, comprising a kind of immune antagonist being provided in addition for patient. The consumption of this immune antagonist cans be compared to its positive usual amounts less (namely using " Asia is the suitableeest " dosage) most. Use inferior dose,optimum be because it may and preparation of the present invention between synergy is arranged. The example of applicable immune antagonist comprises but not only is confined to dexamethasone, imuran, ICAM-1, ICAM-2 or cyclosporin A.

3. treatment nonspecific inflammation

Many inflammatory reactions are because the reaction of " nonspecific defense system " causes, and are to be mediated by the lymphocyte that can not produce immunological memory. This class cell comprises granulocyte and macrophage. As described herein, if inflammation is reaction by the nonspecific defense system to be caused or mediates, or related with the reacting phase of nonspecific defense system, can say that namely this inflammation is the result of non-specific system of defense reaction. Comprise the inflammation of closing with following pathological conditions sample by the example of the inflammation due to the reaction of nonspecific defense system at least in part: ARDS (ARDS) or be secondary to septicemia, wound or the reperfusion injury of hemorrhage multiple organ injury's syndrome, cardiac muscle or its hetero-organization, acute glomerulonephritis, adjuvant arthritis, transfer the toxicity that syndrome and cytokine are induced with other inflammatory diseases of acutely inflamed skin disease, acute purulent meningitis or central nervous system such as apoplexy, fire damage, haemodialysis, leucocyte deformity, ulcerative colitis, regional ileitis, NEC, granulocyte.

Antiinflammatory of the present invention be can the antagonism granulocyte on CD-18 compound and the interactional compound of endothelial cell. This class antagonist comprises: the NIg antagonist of the functional derivatives of ICAM-3, ICAM-3, the ICAM-3 except ICAM-1, ICAM-2 or CD-18 molecule family member.

B. the inhibitor that repels of organ and tissue

Because ICAM-3 particularly its soluble form can work in the mode identical with anti-CD-18 molecule family member's antibody, so can repel in order to the organ or tissue that suppresses to be caused by any cell adherence dependence function. In addition, also can use the functional deriv of anti-ICAM-3 antibody, ICAM-3 and the antagonist of ICAM-3 to suppress this rejection.

Can use ICAM-3 and anti-ICAM-3 antibody to prevent the rejection of parenchymatous organ in the mammal or tissue (such as kidney) or unsubstantiality organ or tissue (such as marrow), perhaps improve oneself immune response. Importantly use the monoclonal antibody that to identify ICAM-3, so as to can between the individuality of HLA mismatch, carrying out organ transplant.

C. the ancillary drug of being convenient to introduce the antigenicity material that uses for treatment or diagnostic purpose

Immune response to some treatment or diagnosticum such as bovine insulin, interferon, tectotype plasminogen activator or little mouse monoclonal antibody can damage treatment or the diagnostic value of these preparations to a great extent, and can cause disease such as serum sickness in some cases. This situation can use antibody of the present invention to treat. In this embodiment of the present invention, can will resist ICAM-3 antibody and treatment or diagnosticum to unite use. Add antibody and can prevent the recipient to the identification of above-mentioned medicament, thereby prevent that the recipient from producing the immune response of anti-these medicaments. Eliminate can make that patient can accept to add after the above-mentioned immune response control malignant boil or diagnosticum.

When being used for the treatment of disease, ICAM-3 (particularly its soluble form) or its functional deriv can use separately or share with ICAM-1 or ICAM-2, or share with the antibody that can be combined with LFA-1. Therefore, can use molecules in inhibiting organ or the graft rejection of this soluble form. In order to reduce the immunogenicity for the treatment of or diagnosticum, can use ICAM-3 or its functional deriv in the mode identical with anti-ICAM-3 antibody.

D. tumour shifts antagonist

Can also use preparation of the present invention need to suppress the transfer of the hematopoietic system cancer cell that the member of CD-18 family participates in. According to this embodiment of the present invention, can give need the patient of this treatment provide the amount that can suppress said transfer completely preparation (if with the toxin of the antibody of ICAM-3 combination, said antibody derive product, said antibody can be in conjunction with the toxin of the fragment of ICAM-3, said fragment derive product, the basically functional deriv of pure ICAM-3, ICAM-3 or the NIg antagonist of the ICAM-3 except ICAM-1 or ICAM-2).

In another example of the present invention, provide the method for the tumour cell growth that suppresses expression ICAM-3. Specifically, the method comprises that patient to this treatment of needs provides the therapeutic agent of q.s. Applicable therapeutic agent comprises toxin derivant, the CD-18 molecule family member's of the antibody that can be combined with ICAM-3, the toxin derivant of said antibody, the fragment that can be combined with ICAM-3 of said antibody, said fragment toxin product or CD-18 molecule family member's the toxin of the functional deriv product of deriving of deriving.

The method of the tumour cell growth that suppresses expression LFA-1 is provided in another example of the present invention. Specifically, the method comprises that the patient to this treatment of needs provides the toxin that can suppress said increment completely. Applicable toxin comprises the toxin derivant of ICAM-3, the perhaps functional derivatives of the toxin derivation of ICAM-3.

E. suppress HIV and infect and prevent the cell diffusion that HIV infects

In another embodiment of the present invention, the method that provides a kind of HIV of inhibition to infect, the method comprises to being subjected to HIV the infected to use the HIV infection inhibitor of effective dose. Suppress the method that HIV-1 infects although the present invention relates to particularly, it should be noted that the method also is applicable to the HIV variant (such as HIV-2) of the in some way infection cell of any available preparation inhibition of the present invention. With regard to purpose of the present invention, these variants are the equivalent of HIV-1.

One aspect of the present invention is based on to be recognized owing to HIV infects the expression that has stimulated LFA-1, and the expression that has stimulated in some cases the LFA-1 binding partner, thereby promoted cell-cell adherence reaction, be increased time of contact of infected cell and non-infected cells, be conducive to virus and shift to non-infected cells from infected cell. Therefore the present invention's preparation that can regulate the LFA-1/ ligand interaction can suppress by HIV the infection that is particularly caused by HIV-1. Suppressing a kind of method that HIV infects by the molecule that suppresses the LFA-1/ ligand interaction is to destroy by the LFA-1 part of the cellular expression of the HIV infection ability in conjunction with the CD11/CD18 acceptor on the healthy T cell. In addition, the molecule that suppresses the LFA-1/ ligand interaction can damage the ability that the CD11/CD18 acceptor by the cellular expression of HIV infection combines with the LFA-1 of healthy T cell. In order to damage cell and the ability that CD11a/CD18 acceptor or LFA-1 binding partner combine, might use the fragment of ICAM-3, ICAM-3, functional derivatives or the anti-ICAM-3 antibody of ICAM-3.

Can preparation of the present invention be offered the recipient by being enough to suppress the amount that HIV infects. In conventional administration situation, if institute is enough to weaken to dosage or stop HIV to infect, can say that namely dosage is enough to " inhibition " HIV and infects. These preparations are provided for contacted HIV or are subject to the patient that HIV infects.

In processing the HIV infection, preparation of the present invention can be used for " prevention " or " treatment " purpose. When being used for prevention, can be before any virus infections symptom occurring (as before infection, during infection or after infecting soon but before any infection symptoms occurring) said preparation is provided. For the prevention administration be for stop or weaken continue after HIV infect. When being used for the treatment of be, can be when detecting the cell that is infected by the virus (or afterwards long-time in) said preparation is provided. The further infection that the treatment administration is intended to alleviate HIV.

Therefore, before preparation of the present invention can be used on virus infections and begins (infecting in order to the HIV that suppresses to occur) or after really detecting by the cell of virus infections (in order to suppress further infection).

In another embodiment, provide improved treating AIDS method, strengthen suppressing the particularly method that infects of HIV-1 of HIV, the method comprises simultaneously and giving:

(I) ICAM-3, solubility ICAM-3 derivative, CD11 (CD11a, CD11b or CD11c), solubility CD 11 derivatives, CD18, solubility CD18 derivative or CD 11/CD18 heterodimer, or the soluble derivative of CD11/CD18 heterodimer, and/or

(II) antibody that can be combined with ICAM-3, and

(III) CD4 that links with cell or particle or the soluble derivative of CD4 and/or

(IV) molecule that can be combined with CD4 better is antibody or antibody fragment.

In another embodiment of the present invention, the method for the cell migration that suppresses the HIV infection is provided, the method comprises the anti-cell migration agent that effective dose is provided to the individuality that infected by HIV.

Migration inhibitor of the present invention comprises the fragment of ICAM-3, the ICAM-3 that can damage the ability that T cell that HIV infects is combined with the LFA-1 part, functional deriv or the anti-ICAM-3 antibody of ICAM-3. Can the ICAM-3 of the T cellular expression by infringement HIV infections be suppressed cell migration with the expression ability that CD11/cell of CD18 acceptor is combined in conjunction with the antibody of ICAM-3. The ability of being combined with the CD11a/CD18 acceptor in order to destroy cell, might use can be in conjunction with the antibody of ICAM-3.

Can provide preparation of the present invention to patient by the amount of the T cell migration that is enough to suppress HIV (or other viruses) infection. If routinely approach administration, dosage can weaken or stop this migration completely, thinks that namely this amount is enough to the migration of " inhibition " T cell.

Can use these compounds for " prevention " or " treatment " purpose. When being used for prevention, can occur before any infection symptoms (for example before infection, when infecting, or after infecting soon but occur before any infection symptoms) said preparation is provided. The prevention administration be for stop or weaken continue after by the migration of the cell of virus infections. When being used for the treatment of, can be when detecting the T cell of virus infections (or afterwards long-time in) said preparation is provided. The treatment administration is be used to weakening the further ability of migration of these T cells.

Therefore, preparation of the present invention can be used on virus infections begin before (in order to the migration of the infected T cell that suppresses to occur) or really be checked through the cell of this virus infections after.

F. treat asthma

In another embodiment of the present invention, can use and can regulate the interactional preparation treatment of LFA-1/ICAM-3 asthma, the method comprises that the individuality to this treatment of needs provides the anti-asthmatic agent of effective dose.

Anti-asthmatic agent of the present invention comprises can damage cell in conjunction with the fragment of ICAM-3, the ICAM-3 of the ability of LFA-1, functional deriv or anti-ICAM-3 antibody of ICAM-3. Suppress the migration of eosinophil by the ability of destroying the ICAM-3 that expresses on these cells and being combined with the cell of expressing the CD11/CD18 acceptor in conjunction with the antibody of ICAM-3.

The consumption of anti-asthmatic agent of the present invention should be enough to reduce or slow down seriousness and the duration of SOA.

Preparation of the present invention can use separately, also can with one or more other anti-asthmatic agents (such as methyl xanthine, theophylline), beta-adrenergic activator (such as catecholamine, Lei Suoxin, saligenin, ephedrine), glucocorticoid (such as hydrocortisone), chromone (such as nasmil) and anticholinergic agents share, to reduce the consumption of preparation of the present invention.

Preparation of the present invention can be used for " prevention " or " treatment " purpose. When being used for prevention, can before occurring, use SOA. The purpose of said preparation prevention administration is intended to prevent or alleviate the SOA of secondary. When being used for the treatment of, must (or thereafter soon) medication when SOA occurs. The said preparation treatment is used and is intended to alleviate any actual asthma attack that occurs. Therefore, preparation of the present invention can use (in order to the order of severity or the shortening duration of seizure of alleviating outbreak) before asthma attack occurring, perhaps use after outbreak.

G. diagnosis and prognosis are used

ICAM-3 in the patient body expresses and the instrument of inflammation part as manifesting can to use the monoclonal antibody that can be combined with ICAM-3. In this application, can carry out the detectability mark by radionuclide, affinity labelling thing (such as biotin, avidin etc.), fluorescein label or paramagnetic atom antagonism ICAM-3 monoclonal antibody. Then the antibody of mark can be used for diagnosis video method. Grossman, H.B. (Urol.Clin. North Amer.13:465-474 (1986)), Unger, E.C. wait people (Invest. Radiol.20:693-700 (1985)) and Khaw, the people such as B.A. (Science 209:295-297 (1980)) have commented the clinical practice of antibody in diagnosis video method.

Also can by the hybridization probe that uses ICAM-3 mRNA sequences in being present in the cell of expressing ICAM-3 or ICAM-3 gene order to be combined, detect the expression of ICAM-3 such as mRNA, cRNA, genomic DNA or synthetic oligonucleotide probe. Maniatis, the people such as T (Molecular Cloning, a Laboratory Manual, Cold Spring Harbor, NY (1982)) and Haymes, the people such as B.D (Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, DC (1985)) technology of finishing this cross experiment described.

The expression that the antibody of usage flag or nucleic acid probe detect TCAM-3 can be used to diagnose tumour. In a diagnosis example, in the checked object body, gather tissue or blood sample, in the presence of its antibody with detectable label, be incubated. In a preferred version, by using magnetic imaging method, fluorescence image Photographic technique etc., finish this technology with the not damaged means. The early stage sign that can utilize this diagnostic test monitoring organ (such as kidney) transplanter to have inorganization to repel. Also might determine whether patient easily suffers from rheumatic arthritis or other chronic inflammatory diseases with this detection method.

For example, by the anti-ICAM-3 antibody of radioactivity mark or antibody fragment, might be with the radioimmunology detectable antigens. The description of relevant radioimmunology (RIA) can be referring to Laboratory Techniques and Biochemistry in Molecular Biology, Work, T.S.et al., North Holland Publishing Company, NY (1978) is visible Chard particularly, and the exercise question that T. writes is " An Introduction to Radi-oimmune Assay and Related Techniques " chapter (this article is classified this specification list of references as). In addition, also available fluorescent chemicals, enzyme or other suitable labels are pressed the known method labelled antibody.

Except determining inflammation part, also can use the antibody that can be combined with ICAM-3 and utilize the culture medium commonly used that is suitable for doing body fluid analysis, whether there is the ICAM-3 of circulation in the detection of biological body fluid. Existing circulation ICAM-3 to show in the body fluid has the biological function of inflammatory reaction or other ICAM-3 mediation. In addition, exist ICAM-3 relevant with the complication of period of gestation in the amniotic fluid. Can carry out this detection to any biological body fluid, but said biological body fluid preferably blood, serum, blood plasma, synovia, amniotic fluid, spinal fluid or urine.

VIII. the medication of the present composition

Use any peptide that therapeutic activity arranged of complete ICAM-3 molecule or its can obtain the result for the treatment of of ICAM-3 to patient. Wherein useful especially is the therapeutic activity fragments of peptides of the ICAM-3 of soluble form.

Available synthetic method, DNA restructuring technology, albumen hydrolysis or share above-mentioned method and make ICAM-3 and functional deriv thereof. Can use ICAM-3 functional derivs with plus Amino Acid residue (in order to improve and the ability of carrier coupling or the activity of enhancing ICAM-3), to improve the result for the treatment of of ICAM-3. The present invention also comprises the functional deriv that lacks some amino acid residue or contain the ICAM-3 of the amino acid residue that changes, as long as these derivatives have biology or the pharmacology activity that maybe can affect ICAM-3.

There is no naturally occurring material if contain in the preparation of ICAM-3 molecule of antibody of the present invention and this paper discussion, can say that namely these antibody and ICAM-3 molecule are " there is no natural pollutant ".

The present invention expands to the antibody (polyclone or monoclonal antibody) that can be combined with ICAM-3, and biological active fragment. These antibody can be produced by animal, tissue culture or recombinant DNA method.

ICAM-3 or can be used separately by it molecule of deriving also can share with ICAM-1 and/or ICAM-2. Anti-ICAM-3 antibody or can also can use separately or share with anti-ICAM-1 antibody and/or anti-ICAM-2 antibody with ICAM-3 or by other molecules that the molecule that it is derived is combined. When using antibody to patient or can be with antibody fragment that ICAM-3 is combined the time, maybe when using ICAM-3 (or its fragment, variant or derivative) to patient, dosage can be different according to the factor such as year the making of patient, body weight, height, sex, general medical situation, medical history. Generally speaking, the dosage of antibody is 1pg/Kg-10mg/Kg (patient body weight) but actual medication also can be below or above this dosage range. When using ICAM-3 molecule or its functional deriv, dosage is more fortunately in 1pg-10mg/Kg (patient body weight) scope, but also can be below or above this dosage range. As discussed below, if will resist ICAM-3 antibody and anti-LFA-1 antibody, anti-ICAM-1 antibody and/or anti-ICAM-2 antibody combined uses, can reduce the effective dose in the treatment. The concept used such as text, when the administration time of two kinds of compounds approaches very much so that can detect this two kinds of compounds simultaneously in the patients serum, i.e. a kind of compound and the second compound associating administration.

The antibody that can be combined with ICAM-3 and the method for administration of ICAM-3 itself can be in intravenous, the muscle, approach in subcutaneous, the intestines, outside part or the intestines and stomach. If antibody or ICAM-3 are administrated by injection, then can be continuous injection, or the single or multiple injection.

The dosage of preparation of the present invention should reach " effective on the physiology " dosage. If the dosage of said preparation and approach etc. are enough to weaken or to stop the associated biomolecule of ICAM-3 to learn effect, then this consumption namely be said to be on the physiology effectively. For example, when using preparation of the present invention to suppress inflammation to patient, can " suppress " inflammation completely such as dosage, then said preparation is exactly effective on the physiology.

In addition, (especially when using to organ or tissue's transplant recipient) can be used separately or be share with one or more other immunodepressant to anti-ICAM-3 antibody or its fragment. These compounds can be used for " prevention " or " treatment " purpose. When being used for the prevention purpose, can before inflammatory reaction or symptom occur, use these immunosupress compounds (for example, can be before organ or tissue transplants, when transplanting or after transplanting soon but the front medication of any organ rejection's symptom occurs) be prevent purpose use these compounds can stop or alleviate any continue after inflammatory reaction (as to the rejection of transplanted organ or tissue etc.). When being used for the treatment of purpose, can (or thereafter soon) medication when inflammatory symptom (such as organ or tissue's rejection) really occurring. Use the purpose of these compounds for therapeutic purposes and be to alleviate any inflammation (such as the rejection to transplanted parenchymatous organ or tissue (such as kidney) or unsubstantiality organ (or marrow)) of actual appearance.

Therefore, antiinflammatory of the present invention can use (being used for the inflammation that inhibition may occur) or use after inflammation occurs before inflammation occurs.

If certain composition can be tolerated by medication person, then said composition namely is " acceptable on the pharmacology ". If its dosage is enough at physiology, can say that then the said preparation dosage is " treatment effective dose ". Change if the existence of certain preparation can detect physiology in medication person's body, then said preparation namely has pharmacological significance.

Can be according to the known method in pharmacy field with antibody of the present invention and ICAM-3 molecule, or its functional deriv combines with the pharmacology acceptable carrier, makes pharmaceutically useful composition. Applicable carrier and prescription thereof (preparation that comprises other people protein such as human serum albumins etc.) are at Remington ' s Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)) is described in. In order to make the pharmaceutically acceptable composition that is suitable for clinical administration, these compositions can contain the preparation of the present invention of effective dose together with the carrier of appropriate amount. In addition, can make antibody humanization of the present invention by chimeric or CDR grafting, be easier to be accepted by human body thereby become. In BP application 900 9548.0, No. 9009549.8 and PCT application PCT/US91/No. 02942 and PCT/US91/02946 number (applying date of accepting office in the U.S. is on April 27th, 1991) method for preparing the anti-especially ICAM-1 antibody of this chimeric antibody has been described.

Can use in addition the pharmacy method control useful effect phase. Can be by control the release of these preparations with polymer compound or absorption preparation of the present invention. Can controlled travelling speed and cycle be adjusted to a specific degree by selecting suitable large molecular matrix and change institute to add macromolecular concentration. Discharging the another kind of possible method of control action time by the control preparation is that preparation of the present invention is incorporated in the particle of polymeric material such as polyester, polyamino acid, aqueous gel, PLA or EVAc. In addition, can also use condensation technique or interface polymerization, utilize gelatin or poly-(isopropyl olefin(e) acid methyl esters) to be embedded in these materials in the microcapsules or be embedded in colloid medicine transportation system for example in liposome, albumin microsphere, microemulsion, molecule and small capsule or the large molecule emulsion.

The present invention also comprises the medical composition that contains following component:

(1) antiinflammatory (if fragment, the functional deriv of ICAM-3, ICAM-3 or the NIg antagonist of the ICAM-3 except ICAM-1 and ICAM-2 of the antibody of being combined with ICAM-3, the said antibody that can be combined with ICAM-3), and (b) at least a immune antagonist. The example of applicable immune antagonist comprises: dexamethasone, imuran and cyclosporin A.

On the basis of the present invention being done general description, now in conjunction with the following example preparation of the present invention and its preparation method are described further, these embodiment are intended to further explain rather than limit the present invention.

Embodiment 1

ICAM-3, the feature of the adhesion part of a kind of new LFA-1

By preceding method clone and lymphocyte are adhered on the coated plate of LFA-1 (Dustin et al., Cold Spring Harbor Symp.Quant.Biol.54:753-765 (1989)). Will be from the JY lysate LFA-1 of purifying with 1100 sites/μ m²Adsorption capacity be adsorbed onto on the 96 aperture microtiter plates (Linbro-titertek). Also use PBS/5%FBS/ 2mM MgCl with 1%BSA sealing nonspecific binding site₂/ 0.5%HSA (detection culture medium) washes each hole. Add the TS1/22 (anti-LFA-1 α) of 1/200 dilution and be incubated 30 minutes under room temperature in microtiter well, the specificity that reaches LFA-1 suppresses. Filter that to separate static T cell from whole blood (be 91%CD2 through plastics absorption and nylon are cotton⁺), simultaneously in the presence of 10 μ g/ml lectins (PHA) cultured cell with generation PHA mother cell. With fluorchrome BCECF (Molecular Probes Inc.) labeled cell, wash to be suspended in again behind the cell and detect in the culture medium. Be incubated 45 minutes 4 ℃ of ascites with cell and 1/200 dilution, so that cell is carried out the MAb preliminary treatment, then in every hole, add 10⁵Individual cell. 37 ℃ of insulations 1 hour make cell adherence to solid phase LFA-1, absorb not adherent cell (6 times) with No. 23 syringe needles, simultaneously are incubated 30 minutes with centrifugal 5 minutes of 30xg with the precipitation lymphocyte and in 37 ℃. Between each washing, with 100 ψ attack culture mediums 8 times, from flat board, remove lymphocyte. So the T lymphocyte that is difficult to the not combination of removing because volume is little can be more effectively removed in attack. Use the fluorescence volume in the direct quantitative analysis 96 hole flat boards of fluorescence concentration analysis instrument (Baxte). All used MAb are saturated concentration (ascites of 1/200 times of dilution), and comprise TS1/22 (anti-CD11a, IgG1) (Sanchen-Madrid et al., Proc.Natl. Acad.Sci.USA.75:7489-7493 (1982)), RR1/1 (anti-ICAM-1, IgG1) (Rothlein et al., J.Immunol.137:1270-1274 (1986)), CBR-1C2/2 (anti-ICAM-2, IgG2a) (de Fou-gerolles et al., J.Exp.Med.Submitted (1991) (waiting to publish)) and CBR-IC3/1 (anti-ICAM-3, IgG1). Make murine myeloma cell P3 * 63Ag8.653 and Balb/c mouse (the Geffer et al. that derives from the SKW3 immunity, Som.Cell Gen.3:231-236 (1977)) splenocyte merges, and suppress the ability that SKW3 is combined with the LFA-1 of purifying according to it and filter out 600 myeloma, thereby make CBR-IC3-1. CRB-IC3/1 performance not with the LFA-1 reaction of purifying, also not with COS cell effect (data are not shown) with ICAM-1, ICAM-2 or LFA-1cDNA transfection. Shown the result of one of four representative experiments, wherein the error bar chart represents a standard deviation.

Embodiment II

The flow cell analysis of accounts that cell ICAM-1, ICAM-2 and ICAM-3 express

Used cell is all as described in the people (J.Clin.Invest.85:674-681 (1990)) such as former Hibbs. Make PMBC (PBMC) by glucan precipitation and Ficoll-Hypaque (1.077) centrifugal process stated. By the red blood cell that reclaims neutrophil and pollute through hypotonic dissolution destruction in the cell precipitation group. Utilize and to carry out the cell count analysis with vertical light scattering forward, so that monocyte is separated with the T cell, and confirm it with monocyte and T cell specificity MAb. With the plastics adhesion method from PBMC lymphocyte-rich and in the presence of 10 μ g/ml lectins (PHA) cultured cell. With EPICS V flow cell counter analytic sample, and use EPICS Immune-Brite fluorescent bead (Coulter) that fluorescence is carried out quantitatively with the calibration cell counter. On the akinete expression of ICAM-3 than CD3 or the large 2-3 of LFA-1 doubly, and the ICAM of monocytes-3 manys 3-4 doubly than LFA-1. The expression of the upper ICAM-3 of neutrophil(e) cell equates with Mac-1 (CD11b/CD18). Processing cell with phospholipase C shows and does not have the ICAM-3 of being combined with PI.

Embodiment III

The immunoprecipitation of ICAM-3

By state method (Kishimoto et al., J.Biol.Chem.264:3588-3595 (1989)) with iodine (¹²⁵I) cell is carried out surface markers. With Triton X-100 (1%) dissolved cell. The Sepharose presettling lysate that adds ox IgG coupling, the Sepharose of then being combined with suitable MAb insulation 2 hours. Wash the sample globule and in the sample buffer that contains 50mM Tris, 1%SDS and 1%2-mercaptoethanol, boil. In the buffer solution that does not contain 2 mercapto ethanol, boil non-reducing sample, and process with 20mM sulphur acetamide. By stating method (Laemmli, U.K., Nature 227:680-685 (1970)) analytic sample on 7% vertical panel polyacrylamide gel, and manifest protein with the autoradiograph method. By former described method (Tarentino et al., Biochemistry 24:4665-4671 (1985)), use certain density N-dextranase (Genzyme) to process sample (10 units/ml, 37 ℃, 18 hours), used concentration is should be able to be from the peptide main chain the most appropriate falls the oligosaccharides that all N connect. I class MHC does not contain the carbohydrate that N connects, and ICAM-2 (molecular weight 600,000,6 N connect site, main chain molecular weight 28,383) then is high glycosylation.

                             Table 2

Determine the relevant surperficial antigen presentation of ICAMs by the immunofluorescence flow cytometry

Clone/type	Specific linear fluorescence intensity *
					αICAM-1                 αICAM-2                  αICAM-3          (RR1/1)                  (CBR-IC2/1)               (CBR-IC3/1)
	5 days PHA-mother cells of static lymphocyte 3 days PHA-mother cells of 1 day PHA-mother cell monocyte neutrocyte		4   22   85   25   13   1	22   18   42   45   39   0					106     78     205     223     224     213
T lymphoblast SKW3 Jurkat Sup T Molt 4					0   2   10   1	98   166   113   242	73     161     19     117
		B lymphoblast JY SLA Ramos Raji		154   224   132   266				119   113   136   88	47     306     112     0
Monocyte U937 HL60					62   17	67   43	37     203
		Melanoma BK RPMI7591		896   475				3   0	0     0
The K562 of erythroleukemia					293	143	0

                             Table 2 (continuing)

Determine the relevant surperficial antigen presentation of ICAMs by the immunofluorescence flow cytometry

Clone/type	Specific linear fluorescence intensity *
					αICAM-1               αICAM-2                αICAM-3          (RR1/1)               (CBR-IC2/1)              (CBR-IC3/1)
	The * * HUVEC Hep G2 HeLa RD3/5 FS1 that mixes, 2,3 A-172		31  1526  1082  0  409  0	494  41    9  0  0					0     0     0     0     0     0

* described in materials and methods, pass through the film expression that the immunofluorescence flow cytometry is determined. Resulting value is the result of at least twice experiment. Calibrate hemacytometer with fluorescent bead, so that 1 unit is approximately 10³Fluorescein equivalent (Coulte Diagnositics, Hialeah, FL).

* mixes clone and comprises: human umblilical vein endothelial cell, huvec; Human breast cancer cell, Hep. G2; People's epithelial cancer cell line, HeLa; The human rhabdomyosarcoma, RD3/5; Human fibrosarcoma, and FS1,2,3; People's glioblastoma.

                         Table 3

By the relevant surperficial antigen presentation of the definite ICAM-3 of immunofluorescence flow cytometry

Clone/type	Than linear fluorescence intensity *
				αICAM-1   (RR1/1)	αICAM-2   (CBR-IC2/1)	αICAM-3   (CBR-IC3/1)
	Static lymphocyte	4	22				106
1 day PHA-mother cell				22	18	78

                             Table 3 (continuing)

By the relevant surperficial antigen presentation of the definite ICAM-3 of immunofluorescence flow cytometry

Clone/type	Than linear fluorescence intensity *
				αICAM-1     (RR1/1)	αICAM-2  (CBR-IC2/1)	αICAM-3  (CBR-IC3/1)
	3 days PHA-mother cells	85	42				205
5 days PHA-mother cells				25	45	223
	Monocyte	13	39				224
Neutrocyte				1	0	213
	The T lymphoblast
SKW3
		0	98	273
Jurkat					2	166	161
	Sup 1	10	115	19
Molt 4					1	242	117
	The B lymphoblast
JY					154	119	47
	SLA	224	113	308
Ramos					132	136	112
	Raji	266	88	0
Monocyte
				U937
	62	67	37
				HL60	17	43	203
MC
				BK	896	3	0

                     Table 3 (continuing)

By the relevant surperficial antigen presentation of the definite ICAM-3 of immunofluorescence flow cytometry

Clone/type	Than linear fluorescence intensity *
				αICAM-1     (RR1/1)	αICAM-2  (CBR-IC2/1)	αICAM-3 (CBR-IC3/1)
	RPMI 7591	475	0				0
Erythroleukemia
				K562	293	143	0
The * * that mixes
				HUVEC	31	494	0
Hep G2	1526	41	0
				HeLa	1082	0	0
RD3/5	0	9	0
				FS1，2，3	409	0	0
A-172	0	0	0
				* described in materials and methods one chapter, determine film expression by the immunofluorescence flow cytometry. Resulting value is the result of at least twice experiment. Calibrate hemacytometer so that 1 unit is approximately 10 with fluorescent bead3Fluorescein equivalent (Coulter Diabnostics, Hialeah, FL) * * mixes clone and comprises: human umblilical vein endothelial cell, HUVEC; Human breast cancer cell, Hep, G2; People's epithelial cancer cell line, HeLa; The human rhabdomyosarcoma, RD3/5; Human fibrosarcoma, and FS1,2,3; People's glioblastoma.

                                         Table 4

ICAM-3 mABs
							PMA-induces		Assemble *
mAb	Homotype	Use separately	+IC1+2mAb	+IC1+2+IC3/1     mAb
					CBR-IC3/2	IgG2a，k	3	2	0
CBR-IC3/3	IgG2a，k	4	3	2
					CBR-IC3/4	IgM，k	4	3	2-3
CBR-IC3/5	IgG2a，k	2	2	0-1
					CBR-IC3/6	N.D.	5	5	5
The ICAM-3 antiserum		3	0	0
					CBR-IC3/1	IgG1，k	5	4	-
HP2/19**			1
					CBR-IC3/2,3/3,3/5 immunoprecipitate 120kK bands of a spectrum are same as the sedimentary bands of a spectrum of CBR-IC3/1. Detect the ability that mAbs forms the Western trace
* refer to the gathering level put down in writing in the J.Exp Med. magazine in 1991. The another kind of ICAM-3 that * obtains from the Spain laboratory. After drawing our result, remind them once to prepare but do not had a kind of mAb of characterization. The result is confirmed that it is ICAM-3.

Claims (40)

1. the method for the variant that substituted by the hydrophilic amino acid residue without the ICAM-3 of natural pollutant or its fragment that lacks the cross-film district or the hydrophobic amino acid residue in the cross-film district of ICAM-3 of preparation, it comprises the said ICAM-3 of recombinant DNA developed by molecule or fragment or variant by can encode described ICAM-3 or fragment or variant, wherein said recombinant DNA molecule comprises a nucleotide sequence, this nucleotide sequence coded at least a following amino acid sequence that is selected from:

A) amino acid sequence of Figure 19 A;

B) amino acid sequence of Figure 19 B;

C) amino acid sequence of Figure 19 C.

2. can encode ICAM-3 or its lacks the preparation method of the recombinant DNA molecule of the variant that the fragment in cross-film district or the hydrophobic amino acid residue in the cross-film district of ICAM-3 substituted by the hydrophilic amino acid residue, comprise DNA sequence clone to a suitable carrier with the described ICAM-3 of coding preparing described dna molecular, the dna sequence dna of the described ICAM-3 that maybe will encode is cloned into a suitable carrier, changes nucleotide sequence with described fragment or the variant that obtains ICAM-3 and the described dna molecular of preparing encode described ICAM-3 fragment or variant;

Wherein said recombinant DNA molecule comprises at least a nucleotide sequence that is selected from lower group:

A) nucleotide sequence of accompanying drawing 19A;

B) nucleotide sequence of accompanying drawing 19B; With

C) nucleotide sequence of accompanying drawing 19C.

3. according to claim 2 method, the amino acid sequence of wherein said recombinant DNA molecule encoding accompanying drawing 19A, 19B and 19C.

4. according to claim 2 method, wherein said recombinant DNA molecule comprises at least a nucleotide sequence that is selected from lower group:

A) nucleotide sequence of accompanying drawing 19A;

B) nucleotide sequence of accompanying drawing 19B; With

C) nucleotide sequence of accompanying drawing 19C.

5. the method for preparing antibody, this antibody can be combined with the molecule that is selected from ICAM-3 or its and lacks the variant that the fragment in cross-film district or the hydrophobic amino acid residue in the cross-film district at ICAM-3 substituted by the hydrophilic amino acid residue, and the method may further comprise the steps:

A) the ICAM-3 immune animal of usefulness purifying,

B) splenocyte and the myeloma cell line with animal merges,

C) hybridoma that forms secretory antibody by the splenocyte that merges and myeloma cell,

D) from above-mentioned hybridoma, screen the required hybridoma that can produce the antibody that can be combined with ICAM-3,

E) separate described hybridoma,

F) cultivate described hybridoma with the antibody of acquisition capacity,

G) the described antibody of purifying;

Wherein said ICAM-3 or fragment or variant are to be encoded by the recombinant DNA molecule that comprises at least a nucleotide sequence that is selected from lower group;

I) nucleotide sequence of accompanying drawing 19A;

Ii) nucleotide sequence of accompanying drawing 19B; With

Iii) nucleotide sequence of accompanying drawing 19C.

6. according to claim 5 method, the combination of wherein said antibody and described molecule has weakened the ability that this molecule is combined with acceptor molecule.

7. according to claim 6 method, wherein said acceptor is LFA-1.

8. according to claim 6 method, wherein said acceptor is Mac-1.

9. according to claim 6 method, wherein said acceptor is p150,95.

10. the method for preparing antibody fragment, this antibody fragment can be combined with the molecule that is selected from ICAM-3 or its and lacks the variant that the fragment in cross-film district or the hydrophobic amino acid residue in the cross-film district at ICAM-3 substituted by the hydrophilic amino acid residue, said method comprising the steps of:

A) mensuration is pressed amino acid sequence or its coding DNA sequence of the antibody of claim 5 preparation,

B) preparation can be expressed the dna molecular of the fragment of described antibody,

C) under the condition that allows described antibody fragment to express, cultivate the cell that transforms with described dna molecular, and

D) the described antibody fragment of purifying.

11. prepare the method for antibody fragment, this antibody fragment can be combined with the molecule that is selected from ICAM-3 or its and lacks the variant that the fragment in cross-film district or the hydrophobic amino acid residue in the cross-film district at ICAM-3 substituted by the hydrophilic amino acid residue, and described method comprises the following steps:

A) proteolysis is pressed the antibody of claim 5 preparation, and

B) the described antibody fragment of purifying.

12. prepare the method for pharmaceutical composition, this pharmaceutical composition is used for regulating the cell biological function of ICAM-3 mediation, wherein said method comprises the ICAM-3 conditioning agent of effective dose is combined with making medicinal diluent, excipient or carrier, and said ICAM-3 conditioning agent is selected from the fragment that can be combined with ICAM-3, ICAM-3 of the antibody that can be combined with ICAM-3, said antibody or it lacks the fragment in cross-film district or the variant that the hydrophobic amino acid residue in the cross-film district of ICAM-3 is substituted by the hydrophilic amino acid residue.

13. method according to claim 12, wherein the biological function of said ICAM-3 mediation is inflammatory reaction, and said ICAM-3 conditioning agent is to provide with the amount that can suppress said inflammation completely.

14. method according to claim 12, wherein said inflammatory reaction is to being selected from the specificity inflammation that occurs in following one group of state response: delayed type hypersensitivity, DTH, psoriasic symptom, autoimmunity disease, organ transplant or the repulsive interaction of tissue transplantation thing.

15. method according to claim 14, wherein said organ transplant is that the parenchymatous organ transplants.

16. method according to claim 15, wherein said organ transplant is kidney transplant.

17. method according to claim 14, wherein said organ transplant right and wrong parenchymatous organ transplants.

18. method according to claim 17, wherein said organ transplant is bone-marrow transplantation.

19. method according to claim 14, wherein said autoimmunity disease is selected from Lei Nuoshi syndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis and lupus erythematosus.

20. method according to claim 14, wherein said inflammatory reaction is the specificity inflammation that forms in to one group pathological state reaction below being selected from: ARDS (ARDS), be secondary to septicemia, wound or hemorrhage multiple organ injury's syndrome, the reperfusion injury of cardiac muscle or its hetero-organization, acute glomerulonephritis, adjuvant arthritis, with acutely inflamed skin disease, acute suppuration arthritis or other inflammatory disease of central nervous system such as apoplexy, fire damage, hemodialysis, the special-shaped disease of leucocyte, ulcerative colitis, Crohn's disease, NEC, granulocyte transfer the toxicity that related syndrome and cytokine bring out.

21. method according to claim 12, wherein the biological function of said ICAM-3 mediation is the transfer of hematopoiesis tumour cell, the functional member who needs CD-18 family during said cell migration, and said preparation is to being to offer said patient's with the amount that can suppress said transfer completely.

22. according to claim 12, wherein the biological function of said ICAM-3 mediation is the leukocytic blood vessel external migration of virus infections, and said ICAM-3 conditioning agent is to offer with the amount that can suppress said leucocyte migration completely to have so leukocytic patient's.

23. method according to claim 22, wherein the cell of said virus infections is infected by HIV.

24. method according to claim 12, wherein said ICAM-3 biological function is the migration of the cell relevant with asthma reaction, and said ICAM-3 conditioning agent is can suppress to offer said patient's with the amount of breathing heavily the relevant cell migration of roaring completely.

25. prepare the method for pharmaceutical composition, this pharmaceutical composition is used for suppressing leucocyte and infected by HIV, wherein said method comprise with the HIV-1 infection inhibitor of effective dose with can make medicinal diluent, excipient or carrier and be combined, derive product, ICAM-3 or its of the toxin that said preparation is selected from the antibody that can be combined with ICAM-3, the toxin derivant of said antibody, the fragment that can be combined with ICAM-3 of said antibody, said fragment lacks the fragment in cross-film district or the variant that the hydrophobic amino acid residue in the cross-film district of ICAM-3 is substituted by the hydrophilic amino acid residue, the member's of CD-18 molecule family member and CD-18 molecule family the toxin product of deriving.

26. method according to claim 25, wherein said HIV is HIV-1.

27. prepare the method for pharmaceutical composition, this pharmaceutical composition is used for suppressing to express the tumour cell growth of ICAM-3, the method comprises the toxin that can suppress said increment completely and can make medicinal diluent, excipient or carrier and be combined, said toxin is selected from the antibody that can be combined with ICAM-3, the toxin of the said antibody product of deriving, the fragment that can be combined with ICAM-3 of said antibody, the toxin of the said fragment product of deriving, and CD-18 molecule family members' the toxin product of deriving.

28. prepare the method for pharmaceutical composition, this pharmaceutical composition is used for suppressing to express the tumour cell growth of LFA-1, said method comprises is combined the toxin that is enough to suppress said increment with making medicinal diluent, excipient or carrier, said toxin is selected from the variant that the ICAM-3 fragment that lacks the cross-film district that ICAM-3 that toxin derives and toxin derive or the hydrophobic amino acid residue in the ICAM-3 cross-film district are substituted by the hydrophilic amino acid residue.

29. each method according to claim 12-28, wherein said method comprises that also use is selected from one or more preparations in following a group: the antibody that can be combined with LFA-1, the fragment that can be combined with LFA-1 of said antibody, can with the antibody of ICAM-1 combination, the fragment that can be combined with ICAM-1 of said antibody, the antibody that can be combined with ICAM-2, the fragment that can be combined with ICAM-2 of said antibody.

30. each method according to claim 12-28, wherein said pharmaceutical composition approach administration in enteron aisle, outside the intestines and stomach, in local, suction or the nose.

31. each method according to claim 12-28, wherein said pharmaceutical composition is used for the prevention purpose.

32. each method according to claim 12-28, wherein said pharmaceutical composition is used for the treatment of purpose.

33. method according to claim 30, wherein said intestines and stomach external administration approach be in the muscle, intravenous or subcutaneous administration.

34. method according to claim 12, wherein said pharmaceutical composition also contain immunodepressant in addition.

35. according to claim 12,25,27,28 or 29 method, wherein said pharmaceutical composition is combined with at least a other immunodepressant.

36. according to claim 25,27 or 28 method, wherein said toxin is selected from lower group: ricin toxin, cholera toxin, diphtheria toxin, radionuclide toxin and film passage form toxin.

37. prepare the method for diagnosing composition, it may further comprise the steps:

A) the detectability mark is pressed the prepared antibody of claim 5 or is pressed the prepared antibody fragment of claim 10;

B) the described antibody of purifying or antibody fragment from the mark mixture; With

C) described antibody or antibody fragment are mixed with diluent, excipient or carrier.

38. detect the method that whether has the cell of expressing ICAM-3, the method comprises:

(1) extract of the said cell of insulation or said cell in the presence of a kind of nucleic acid molecules, said nucleic acid molecules can and comprise at least a nucleotide sequence that is selected from lower group with ICAM-3mRNA hybridization:

A) nucleotide sequence of accompanying drawing 19A;

B) nucleotide sequence of accompanying drawing 19B, and

C) nucleotide sequence of accompanying drawing 19C; With

(2) determine described nucleic acid molecules whether with the extract of described cell or described cell in the complementary nucleic acid molecule that exists hybridize mutually.

39. detect the method that whether has ICAM-3 in the mammalian body fluid sample, the method comprises:

(a) the biological humoral sample of said diagnosis object is incubated with containing the antibody that can be combined with ICAM-3 or the composition of its fragment, and

(b) detect the antibody of being combined with said body fluid.

40. method according to claim 39, wherein said body fluid is selected from blood, serum, blood plasma, synovia, amniotic fluid, cerebrospinal fluid or urine.

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