CN1069522A - ICAM-3 and part thereof - Google Patents

Abstract

The present invention relates to intercellular adhesion molecule (ICAM-3), this molecule is participated in lymphocyte identification and is moved to inflammation part, and in the process that is attached between inflammatory phase on the cell matrix.The present invention relates to like this some molecules, identify these molecules screening method and can with these molecule bonded antibody.The invention still further relates to adhesion molecule and the treatment and the diagnostic use of bonded antibody with it.

Description

ICAM-3 and part thereof

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

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

The invention still further relates to independent use ICAM-3 or unite use, to suppress the intercellular adhesion of granulocyte, lymphocyte or scavenger cell pedigree with ICAM-1 and/or ICAM-2.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 the AIDS diseases such as (acquired immune deficiency syndrome (AIDS)) that is for example caused by HIV virus.

The invention still further relates to independent use ICAM-3, or unite and use dead and " synplasm " 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, as the AIDS (acquired immune deficiency syndrome (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 can with ICAM-3 bonded molecule (hereinafter referred to as anti-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 can with ICAM-3 bonded antibody, peptide or carbohydrate.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 scavenger cell pedigree.Use these molecules that the method for treatment 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 particularly treatment and the prevention method of HIV-1 infection.Therefore it can be used for treating disease, as the AIDS (acquired immune deficiency syndrome (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, with the cell that suppresses the HIV-1 infection by the methods of treatment of moving in the recycle system.Therefore it can be used for treating disease, as the AIDS (acquired immune deficiency syndrome (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. white corpuscle adheres to and function

In order to make the host suitably resist the intrusion of external pathogenic factors such as bacterium or virus, white corpuscle must attached on the cell matrix (referring to Eisen, H.W., Microbiology, 3rd Ed., Harper; Row, Philadelphia, PA(1980), pp.290-295and 381-418).White corpuscle must be attached to moving to inflammation part from circulation on the endotheliocyte.In addition, they must be attached to antigen and present on the cell normal specific immune response could take place.At last, they must be attached to suitably dissolving cell or the tumour cell that is subjected to virus infection on the 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)).The molecule of being identified by these antibody is called as Mac-1 and leucocyte function-associated antigen-1(LFA-1).Mac-1 is the heterodimer that sees on scavenger cell, 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, promptly p150.95(it have the tissue distribution similar to Mac-1), all in cell adhesion, work (Keizer, G.et al., Eur.J.Immunol., 15: 1142-1147(1985)).

Found that above-mentioned white corpuscle 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 " the CD-18 family " of glycoprotein.This glycoprotein family is made up of the heterodimer with a α chain and a β chain.Though 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.Though the α subunit of membranin is not shared by the total extension homology of β subunit, recently the analysis revealed of glycoprotein α subunit had substantial similarity between them.Sanchez-Madrid, people such as F. (J.Exper.Med.158: 586-602(1983); J.Exper.Med.158: 1785-1803(1983)) α of IFA-1 associated glycoprotein and the similarity 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 on its leukocyte surface, express any member of this attachment proteins family with normal amount; 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)).The sex expression of LAD patient characteristics comprises that soft tissue injury, vomica form and wound is difficult for healing, and external adhesion dependency leukocyte function is unusual and to chronic and recidivity infectation of bacteria sensitivity.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 endotheliocyte.But the more important thing is, observe these patients because its granulocyte can not produce normal inflammatory reaction attached to the ability on the cell matrix.The most significant be observe since these patients' LAD granulocyte not attached near the ability on the vascular endothelial cell at inflammation damnification position, so can not reach inflammation part such as skin infections position.This adhering to is the steps necessary of soaking into.

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 cell 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 way owing to lacking the functional adhesion molecule of CD-18 family, i.e. performance has slowed down immune response.

In a word, white corpuscle is for keeping animal health and survival ability, and they must stick on other cells (as endotheliocyte).Found that this adhesive attraction needs cell-cells contacting that the specific receptors molecule on the leukocyte surface is participated in.These are subjected to physical efficiency to make leukocyte adhesion on other white corpuscles, endotheliocyte 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 white corpuscle lacks these cell surface receptors easily suffers from chronic and the recidivity infection, and other clinical syndromes 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 (as kidney) transplanting, unsubstantiality organ (as marrow, tissue) transplanting, transformation reactions and oncology, 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 by virus protein (being called " gp120 ") to be present in T4(" T is auxiliary " that HIV infects) acceptor molecule (being called " CD4 ") on the lymphocytic cell surface goes up the (Schnittman that takes place, S.M.et al., J.Immunol.141: 4181-4186(1988), the document is classified this paper reference as).After being attached on this receptor, virus promptly enters cell and duplicates, 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 infection suffers damage.Usually become tumour patient though infected the people of being grown disease, the relation between these tumours and HIV infect in most of the cases it be unclear that.

Though HIV virus duplicate for infected cells to be lethality, only in the infected person's of sub-fraction T4 cell, to detect this duplicating.Nearest result of study prompting, the incidence of viremia is than the height of estimating in the past, and T cell infection frequency can be up to 1%.

The virus-mediated T4 of HIV colony other mechanism of destructive have been illustrated in several research work.

Except HIV duplicates, 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 (as the blood transfusion of mispairing or organ transplantation etc.).After infected by HIV, the T cell presents the gp120 molecule on its cell surface.Killer cell is external cell (rather than intrinsic cell) with these T4 cell recognition, and then destroys it.

HIV infects the destruction that also can cause the healthy cell of uninfection.Infected cell can be secreted gp120 protein in blood system.Free gp120 molecule just can be attached on the CD4 acceptor of healthy cell of uninfection then.This combination can make cell present the barment tag of the cell of HIV infection.The cellular toxicity killer cell is discerned 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 necrocytosis." syncytial cell " is the giant cell that a plurality of nuclears are arranged, and forms by reaching a hundreds of T4 cytogamy.Make infected cells obtain the ability that merges mutually with other T4 cells (comprising HIV healthy cell that infect or uninfection) after the infected by HIV.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-cells contacting of T4.The cell of HIV infection forms plasmodial ability and shows that these cells need a kind of means that merge with healthy cell.

As if HIV infects, and particularly HIV-1 infects, influence the cell surface expression of leucocyte integrin (integrins), and by the cell adhesion 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 reference 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 endotheliocyte of IL-1 stimulation with the U937 cell than uninfection with bigger frequency; This behavior can handle infected cell with anti-CD18 or anti-CD11a monoclonal antibody or with anti-ICAM-1 antibody treatment endotheliocyte 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 phytohemagglutinin (PHA) stimulation and the synplasm of the negative T cell of infected in fact CD4 forms (Hildreth, J.E.K., et al., Science 244: 1075(1989)).Found to have only the virus infected cell of handling with anti-CD18 or anti-CD11a monoclonal antibody that synplasm is formed almost not influence, this point points out these antibody can protect the target cell that does not infect to avoid infecting (Hildreth basically, J.E.K.et al., Science 244: 1075(1989); Valentin, A.et al., J.Immunology 144: 934-937(1990)).Recently, people such as Valentin (J.Immunology 144: 934-937(1990)) have further confirmed above-mentioned observations, find when with successive T clone and HIV-1 infection U937 co-culture of cells, can suppress synplasm to the special monoclonal antibody of CD18 and form.

Though the mechanism that the cell that CD18 or CD11a monoclonal antibody specific protection sensitive cells avoid infecting with HIV takes place to merge it be unclear that; and relevant mechanism the present invention neither be necessary for estimating; but with the research prompting that radiolabeled gp120 did; 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 infection to pass through the endothelial barrier conveying.Virus-the cell interaction of analog cell-cell interaction can help free virus being adhered to and/or infecting healthy cell.

Therefore, the present invention is based on HIV to a certain extent and infects HIV-1 particularly and infect this observations 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 adhering to each other or accumulative ability (" homotypic aggregation " promptly takes place) of T cell that HIV infects.Because not seeing between the normal white corpuscle of immobilized has 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 takes place, 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 have only the 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 help 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 the leukocytic migration and the distribution of virus infection.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 Microscopic examination showed of impaired organ has the outer monocytic infiltration of local vascular.Attempt to identify the virus infected cell that soaks in the central nervous system, found that the cell that exists HIV-1 to infect in the instillation.These studies show that HIV-1 virus mainly is present in monocyte and the scavenger cell, 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 to stimulate the monocytoid cell of HIV-1 infection to form the outer mechanism of soaking into of blood vessel in the past.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 helps white corpuscle and adheres on vascular endothelial cell, and help white corpuscle and from blood vessel, transfer to extravascular tissue position (C.W.Smith et al., J.Clin.Invest.82: 1746(1988), list this paper reference in), so existing people proposes to use the antibody that suppresses cell migration to prevent the diffusion (WO90/13316) of the cell of HIV infection.

D. asthma: Clinical symptoms

Asthma is an allogenic disease family, it is characterized in that segmental bronchus to stimulator have hyperergy (Kay, A.B., Allergy and Inflammation, Academic Press, NY(1987); This article has been classified this paper reference as).Clinically, as seen there is segmental bronchus generally narrow, heavy-gravity secretory product is arranged, patient's signs such as sound that paroxysmal dyspnea occurs, cough and stridulate.Though also do not know the relative consequence of these symptom, the long and is that aeration resistance increases, and lung and chest level expand, and the unusual distribution of ventilation and lung blood flow.Between this disease shows between asymptomatic stage or acute stage symptom occurs.The oxygen supply deficiency can take place and can cause death in acute phase.Whole world crowd has 3% to suffer from asthma approximately.

Two types asthma has been described: atopic asthma and atopic asthma.Atopic asthma is usually relevant with heritable allergic disorder such as rhinitis, urticaria, eczema etc.Be characterised in that appearance to hypodermic airborne transmission antigen (as pollen, environment and the professional pollutent etc.) wheal-flare reaction that is negative, and serum IgE level occur and increase.Many patients develop into atopic asthma on the cause of disease as and if exist IgE antibody relevant.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 by airborne transmission antigen with the interaction that forms the IgE molecule in advance that combines mastocyte activated.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 factors, occupational factor, manual work and factor such as excited increases the weight of because of capacity.

Can treat by antagonist (as coromegine) with methyl xanthine (as theophylline), Beta-3 adrenergic agonist (as catecholamine, Resorcinol, saligenol and racephedrine etc.), glucocorticosteroid (as hydrocortisone), mast cell degranulation inhibitor (being chromone) and choline as Sodium Cromoglicate.

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

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.Though 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 pathology characteristics of asthma are a large amount of infiltrations of eosinophil to 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 segmental bronchus.According to this hypothesis, eosinophil attracted on the segmental bronchus, takes off particle here to discharge the cellular toxicity molecule.After taking off particle, eosinophil discharges enzyme, as histaminase, aryl sulphatase and phosphatidase D in order in the enzymatic and harmful mediators of mastocyte.Yet these molecules also promote the mucomembranous cilium disorganization, thus overslaugh to the removing of bronchial secretion and cause the injury of lung characteristic of asthma to change.

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

The present invention is based on and has found to be called intercellular adhesion molecule 3(ICAM-3) new cell adhesion molecule.The invention still further relates to the functional derivatives of ICAM-3, anti-ICAM-3 antibody, become 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 the diagnosis and the treatment application of all above-mentioned molecules.

Specifically, the present invention includes ICAM-3 or its functional derivatives that does not have natural pollutant basically.

The invention provides acquisition and can encode or express the method for the molecule of the reorganization 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 said monoclonal antibody.

The present invention includes the method that produces required hybridoma, said hybridoma can produce can with ICAM-3 or its functional derivatives bonded antibody, this 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 immunogen immune animal of the peptide fragment of the peptide fragment of carrier-bound ICAM-3, ICAM-3 or the ICAM-3 that combines with carrier,

B) will merge from isolating splenocyte in the said immunized animal body and myeloma cell,

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 significant quantity to the object of this processing of needs, wherein said ICAM-3 conditioning agent be selected from can with ICAM-3 bonded antibody, said antibody can with functional derivatives and the NIg antagonist except that ICAM-1, ICAM-2 or CD-18 molecule family member of ICAM-3 bonded fragment, ICAM-3, ICAM-3.

The present invention also provides the method for treatment people and other mammiferous specificity inflammation, said method comprise to the object of this treatment of needs use can the inflammation-inhibiting amount anti-inflammatory agent, wherein anti-inflammatory agent be selected from can with ICAM-3 bonded antibody, said antibody can with ICAM-3 bonded fragment, the NIg antagonist of pure in fact ICAM-3, the functional deriv of ICAM-3 or ICAM-3, wherein said inflammation are transplanted (as kidney), unsubstantiality organ transplantation (as marrow) or tissue transplantation by the parenchymatous organ and are caused.

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

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: grownup's respiratory distress syndrome; Be secondary to multiple organ injury's syndromes of septicemia, hemorrhage or wound; The reperfusion injury of cardiac muscle or its hetero-organization; Acute glomerulonephritis; Reactive arthritis; With acutely inflamed tetter; Acute purulent meningitis or other inflammatory disease of central nervous system are as apoplexy; Thermal damage; Hemodialysis; The special-shaped disease of white corpuscle; Ulcerative colitis; Crohn disease; Necrotizing enterocolitis; Relevant syndromes of granulocyte infusion and cytokine inductive toxicity.

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, wherein said preparation be selected from can with ICAM-3 bonded antibody, the toxin of the said antibody derived products of being correlated with, said antibody can with ICAM-3 bonded fragment, the derivative of said fragment and toxin, the functional derivatives of ICAM-3, the toxin of the functional derivatives of the said ICAM-3 derived products of being correlated with, or the NIg antagonist of the ICAM-3 except that CD-18 molecule family member.

The present invention also comprises the method for the growth of tumour cell 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 be selected from can with ICAM-3 bonded antibody, the derived products of said antibody and toxin, said antibody can with the derived products of ICAM-3 bonded fragment, said fragment and toxin, the toxin deutero-member of CD-18 molecule family or CD-18 molecule family member's toxin deutero-functional derivatives.

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 culturing cell or cell extract in the presence of the nucleic acid molecule of ICAM-3mRNA hybridization, and

B) detect and the nucleic acid molecule 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 this method comprises:

A) with can being incubated with said sample with ICAM-3 bonded antibody or its fragment, and

B) detect said antibody and whether combine said sample.

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

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

Fig. 1 showed cell system (A) and lymphocyte (B) are to the adhesion of the LFA-1 of the purifying represented by ICAM-1, ICAM-2 and ICAM-3.(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 microtiter well of IFA-1 bag quilt.Control wells LFA-1 bag of no use quilt.

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

Fig. 3 shows the immunoprecipitation of ICAM-3.(A) the non-binding contrast X63 immunoprecipitation anti-ICAM-3 of usefulness MAb CB-IC3/1() ¹²⁵The cellular lysate of the anti-mark of I; (B) with (+) or need not (-) N-glycanase handle, and with the non-binding MAb of contrast X63, the anti-HLA-A of MAb W6/32(, B, C), the anti-ICAM-1 of MAb RR1/1(), the anti-ICAM-2 of MAb CBR-IC2/1() or the anti-ICAM-3 of MAb CB-IC3/1() immunoprecipitation ¹²⁵The SKW3 cellular 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.Through the isolating ICAM-3 of polyacrylamide gel electrophoresis analysis and use different antibody to carry out the Western engram analysis.

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

Fig. 6 shows the SKW3 cell adhesion situation that antibody blocking and PMA stimulate.By the SKW3 cell of the various antibody blocking PMA stimulations of former described method test and the ICAM-1 or the ICAM-3 bonded ability of purifying.

Fig. 7 shows the situation that combines of COS cell transfecting body and the ICAM of purifying.By former described method LFA-1 expression vector rotaring redyeing COS cell.Detect transfectional cell then when adding or not adding anti-LFA-1 antibody (TS1/22) and immobilization ICAM-1 and ICAM-3 bonded ability.

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

T cell and the influence of ICAM-3 bonded that Fig. 9 displays temperature stimulates PMA.17,22 and 37 ℃ of abilities that detect the T cell of PMA stimulation in conjunction with the ICAM-3 of purifying down.

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

Figure 11 shows the influence of anti-ICAM antibody to the blended lymphocyte reaction.Ability by the various antibody blocking blended of former described method test lymphocyte reaction.

Figure 12 shows the gas chromatography result of ICAM-3 peptide fragment.Purifying also digests ICAM-3 with Lys-C.Analyzed peptide fragment by former described method through high performance liquid chromatography.

Figure 13 shows clone 11.2 cleavage map.NK10 and NK17 peptide, the various dna sequence dnas that so obtain and the position of striding the film district have wherein been pointed out.

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

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. hold the sequence that obtains from 5 of ICAM-3 '.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 the NK-10 probe and obtain.

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

The present invention is based on and found still unidentified and LFA-1 bonded part in the past.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 various kinds of cell such as leukocyte adhesion molecule LFA-1 mediated leucocytes, monocyte, natural killer cell and granulocyte and other cells.

LFA-1 be ICAM-1, ICAM-2 and the ICAM-3(that identifies recently as disclosed herein) acceptor.These surface moleculars are expressed and (Cell 51: 813-819(1987) for Marlin, S.D.et al. by being induced generation on its hetero-organization in inflammatory process 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, February 7 1987 440(applying date) and u.s. patent application serial number 07/250, September 28 1988 446(applying date), these two parts of patent documentations are all classified this paper reference as).

LFA-1 (the springer that in the interaction of antigen-specific 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 waits 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 that (Immunol.Today 9: 213-215(1988) for Dustin, M.L.et al. for the member 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 comprised at an easy rate, and the cytokine of IFN-g, TNF and IL-1 induces, and produces on the number of different types cell (Immunol.Toady 9: 213-215(1988) for Dustin, M.L.et al.).On endotheliocyte, epithelial cell and inoblast, induce the ICAM-1 of generation can mediate the lymphocytic adhesion of LFA-1 dependency (Dustin, M.L.et al., J.Immunol.137: 245-254(1986); Dustin, M.L.et al., J.Cell.Biol.107: 321-331(1988); Dustin, M.L.et al., J.Exp.Med.167: 1323-1340(1988)).Can block with LFA-1 MAb primed lymphocyte or after with other cells of ICAM-1 MAb pre-treatment adhesive attraction (Dustin, M.L.et al., J.Immunol.137:245-254(1986); Dustin, M.L.et al., J.Cell Biol.107:321-331(1988); Dustin, M.L.et al., J.Exp.Med.167:1323-1340(1988)).The result who identifies with the ICAM-1 of purifying in artificial rust or on the plate proves, LFA-1 and ICAM-1 each other acceptor (Cell 51: 813-819(1987) for Marlin, S.D.et al.; Makgoba, M.W.et al., Nature 331: 86-88(1988)).For the sake of clarity, they are called " acceptor " and " part " herein.U.s. patent application serial number 07/045,963,07/115,798,07/155,943,07/189,815 or 07/250, all these patent application documents of 446(are all classified in full this paper reference as) in 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)).Second kind of LFA-1 part being identified named is " ICAM-2 ".

ICAM-2 is in cell distribution and lack cytokine and be different from ICAM-1 aspect inducing two.ICAM-1 is the conformity membrane albumen 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 in two N-terminal zones of ICAM-1, has proved that the subfamily of class Ig part combines same conformity gene family receptors.In the u.s. patent application serial number 07/454,294 ICAM-2 has been done further description (this paper reference 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 observed LFA-1 dependency, ICAM-1 dependent/non-dependent phenomenons in the past, and there is the third part of LFA-1 in prompting.Unite combining of the IFA-1 that uses ICAM-1 and ICAM-2 MAb can block several types cells such as epithelial cell and endotheliocyte and purifying fully, comprise that many lymphoidocytes of t cell lymphoma clone SKW3 are fastened ICAM-1, the ICAM-2 dependent/non-dependent adhesion approach (Fig. 1) that then has at LFA-1.

In order to illustrate this adhesion approach, produced and anti-ICAM-1 and the corresponding to anti-SKW3 MAb of 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, promptly CBR-IC3/1 can suppress this new adhesion approach (Fig. 1) fully.Unite and use anti-ICAM-1 of barrier and anti-ICAM-2 MAb can only slightly suppress the adhesion of SKW3 the LFA-1 of purifying.As the anti-ICAM-3 MAb(CB-IC3/1 of independent adding) time can suppress significantly to adhere to, and can reach inhibition fully when share with the anti-ICAM-2 MAb of barrier.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.Curve proved as suppressing according to the difference of MAb, and 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 the 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 clone Jurkat has used ICAM-2 and ICAM-3 approach when adhering to, and ICAM-1 also has less contribution.Also studied static T lymphocyte and with the lymphocytic adhesive attraction of T (Figure 1B) of phytohemagglutinin (PHA) activation.The static lymphocyte that studies show that in the past can combine (Dustin et al., Nature 341: 619-624(1989)) forcefully with the LFA-1 of purifying, 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 activated T cells,, but still can detect though adherent ICAM-2 composition is covered because of the existence of ICAM-1 and ICAM-3.Because for every kind of cell, need the antibody of anti-two kinds of ICAM at least 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 three kinds of anti-ICAM MAb and get rid of and the combining of LFA-1, and few with the level error that in the presence of anti-LFA-1 MAb, is reached.

Can compare three kinds of ICAM in conjunction with the contribution (detecting) of LFA-1 and their the cell surface expression situation that records with the immunofluorescence flow cytometry, with the relative affinity (Fig. 2) of definite three kinds of ICAM to LFA-1 as top.Relatively the surface expression of ICAM and they are disclosed ICAM-1 LFA-1 are had bigger affinity the adherent contribution of LFA-1, and ICAM-2 and ICAM-3 then have similar but lower affinity.For example, the Jurkat cell can be expressed ICAM-and ICAM-3 with similar level, and separately to making contributions with combining of LFA-1.When ICAM-2 expresses expression greater than ICAM-3 (as on the JY cell), the adherent ICAM-2 approach of LFA-1 promptly 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 understandable fully, because adherent ICAM-3 approach is better than the ICAM-2 approach.ICAM-3 can well be expressed on 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 (as under the situation of SLA, JY and PHA activated T cells), just ICAM-1 has become adherent main path.

The distribution of ICAM-3 is different from ICAM-1 and ICAM-2 in several modes.Different with ICAM-1 and ICAM-2, ICAM-3 does not express (data is not shown) on static or activated endotheliocyte.This point conforms to following discovery: promptly to combine with the LFA-1 dependency of the endotheliocyte of static and irriate be a kind of ICAM-1 and ICAM-2 dependency phenomenon to cell.ICAM-3 is only limited to hematopoietic cell lineage, removes a few exceptions, and it can be fastened highly at lymphoidocyte and monocyte and be expressed.But in all cases, the expression of ICAM-3 on clone is all consistent with adherent LFA-1 dependency, ICAM-1, ICAM-2 dependent/non-dependent approach.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 producing faint bonded clone (JY, U937, SupT) 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 white corpuscle, it obviously is different from ICAM-1 and ICAM-2(Fig. 2 B).ICAM-3 can express on 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 have only ICAM-2 to be present on the static lymphocyte.ICAM-1 and ICAM-2 all do not express on the neutrophil(e) cell.After with the PHA activated lymphocyte, ICAM-3 expresses has increased 2-3 doubly, and ICAM-1 expresses and then is subjected to inducing significantly (Dustin et al., J.Immunol.137: 245-254(1986)) (Fig. 2 B).

Derive from various ¹²⁵The ICAM-3 immunoprecipitate of the clone of I mark presents the clear band of a molecular weight about 124,000 under reductive condition, only increased mobility (Fig. 3 A) a little in non-reduced condition simultaneously.Handle the band (Mr87,000) that the back produces the ICAM-3 of lower molecular weight with the N-glycanase, 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 sticking property all are different from, comprise the people acceptor LAM-1(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 waits to publish); Osborn et al., Cell 59: 1203-1211(1989)) and matrix acceptor (Hemler, the M.E. of VLA family, Ann.Rev.Immunol.8: 365-400(1990)), and the non-MAb(Gilks that sees the similar cell distribution of tool in the Sibai cell database, W.R., et al., Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England(1990)).

There is specialization of this true prompting of three kinds of LFA-1 parts to the different aspect of LFA-1 dependency white corpuscle interaction.ICAM-1 expresses on endotheliocyte and many type epithelial cells, and under inflammation and immune condition, be subjected to inducing by force, to regulate cellular localization and to help specific antigens identification (Wawryk et al., Immunol.Rev.108: 135-161(1989)).Because ICAM-2 is the main LFA-1 part on the static endotheliocyte, so should carry out normal recirculation significant (Hamann et al., J.Immunol.140: 693-699(1988) for the lymphocyte that carries LFA-1 by organizing endotheliocyte by the adhesion approach; 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 parts on monocyte and static lymphocyte, and these are found and the fact shows that it has vital role in starting immune response.The adhesion that T cell and antigen present cell needs 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 ICAM-1 and ICAM-2 well on the type cell.Really, in allos and AMLR, prompting is LFA-1 part rather than ICAM-1 (Bagasco et al., Cell Immunol.128: 362-369(1990)) in action.In addition, infer that it should be very important that the antigen-specific of ICAM-3 between T and bone-marrow-derived lymphocyte interacts, because wherein a kind ofly be not activated as yet 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 analogue 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: the 533-534(1991)) homotransplantation.Because can suppressing the LFA-1 dependency of different subgroup cell types, ICAM-3 MAb interacts, so can be used to suppress specific and immune responses many intersections in vivo.

The cDNA clone of V .ICAM-3

Available several different methods is cloned the ICAM-3 gene.Wherein a kind of method is to analyze the shuttle vectors library of cDNA inset (being derived from the ICAM-3 express cell), with existing of the inset of determining to contain the ICAM-3 gene.The available support transfectional cell detects the expression of ICAM-3 then, to carry out this analysis.

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

As wrapping by plate, can in plate, add anti-ICAM-1 and anti-ICAM-2 specific antibody, to prevent the adhesion of ICAM-1 or ICAM-2 express cell with LFA-1.Available antibodies like this such as the anti-ICAM-1 MAb of RR1/1() and the anti-ICAM-2 MAb of CBR-1C2/2() inhibition ICAM-1 ⁺Or ICAM-2 ⁺Transformant adheres on the plate of LFA-1 bag quilt.The ICAM-3 cells transfected can be suppressed by EDTA and anti-LFA-1 MAb with the combining of plate of LFA-1 bag quilt, but can not be suppressed by anti-IACA-1 or anti-ICAM-2 MAb.Therefore ICAM-1 or ICAM-2 express cell can not adhere on the plate, thus great majority can be washed off with every other non-adherent cell, thereby enrichment the cell of expression ICAM-3.

Other selected for use methods that obtain the gene order of coding ICAM-3 are as known in the art, and those skilled in the art can select for use wherein a kind of method to obtain 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 this 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 aminoacid 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 combined preparation degeneracy probe.Screen the genome or the cDNA library of its sequence and probe hybridization then with this probe.

Also can use genome obtain the encoding cDNA clone (Watson of ICAM-3 in addition, 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 peptide fragment aminoacid sequence of determine to encode ICAM-3 protein or ICAM-3 protein.Use then this probe in detecting cDNA library (from what separate from the mRNA of ICAM-3 express cell preparation) in coding proteinic member of 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)), human estrogen acceptor (Walter, P.et al., Proc.Natl.Acad.Sci.USA 82: 7889-7893(1985)) and (Pennica of tissue-type plasminogen activator, D.et al., Nature 301: 214-221(1983)) gene.

In the another kind of method of clone ICAM-3 gene, can derive from the genomic dna of the cell of expressing ICAM-3 through the clone, be more preferably cDNA and prepare expression library.Screen the proteinic member that can express in the library then with anti-ICAM-3 antibodies.

The clone's that makes by above-mentioned either party's method ICAM-3 gene can be operably connected on the expression vector, and import in bacterium or the eukaryotic cell to produce ICAM-3 protein.Gene manipulation techniques can be referring to Maniatis, 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, agonist and antagonist

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

The functional derivatives of A.ICAM-3

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

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

" varient " of ICAM-3 is meant on the 26S Proteasome Structure and Function basically and whole molecule or the similar molecule of its fragment.

If two molecules have similar basically structure or biologic activity, we can say that then a molecule " similar basically " 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 varient.

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 solvability, adsorptivity of molecule and biology half life etc.This part also may reduce the toxicity or the elimination of molecule or weaken harmful pair of effect of molecule.Remington ' s Pharmaceutical Sciences(1980) molecular moiety that can mediate this effect is disclosed in.

" toxin deutero-" molecule has constituted special " chemical derivative " of a class." toxin deutero-" molecule is the molecule (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, impels necrocytosis thereby can make toxin moiety more approach cell.Can use any suitable toxin moiety; But better be to use ricin toxin, Toxins,exo-, cholera, diphtheria toxin, radionuclide toxin or membrane channel to form toxin.

Can use conventional synthetic method to have the nearly ICAM-3 functional derivatives of 100 residues in external preparation.In case of necessity, available currently known methods is modified such fragment, comprises making organic chemistry modifier reaction purifying or rough proteinic target amino acid residue and the side chain that can select together or terminal residue reaction.Can use the gained covalence derivative to identify that those are for the very important residue of biologic activity.

Can produce and separate the fragment of ICAM-3 in many ways.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) 3,969,287,3,691,016,4,195,128,4,247,642,4,229,537 and 4,330 of cyanogen bromide-activated, the reactive matrix described in 440 fixes protein.

The functional derivatives that also can prepare the ICAM-3 that has changed aminoacid sequence by the DNA of sudden change coding ICAM-3.For example, such functional derivatives comprises the forms such as disappearance, insertion or replacement of residue in the aminoacid 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 and read outside the sign indicating number, and preferably can not cause and will produce the complementary region (referring to EP patent application publication number 75,444) of secondary mRNA structure.

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

Though can pre-determine the site of introducing sudden change in aminoacid sequence, sudden change itself does not need to pre-determine.For example, to reach best effect in order making to the sudden change on the locating 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 the required activity of best of breed.The technology that causes sudden change on the predetermined site in known dna sequence is known, and for example the specificity mutafacient system 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 site-directed 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 reference as.Site-directed mutagenesis allows us to produce the functional derivatives of ICAM-3 by the specific oligonucleotides sequence of the dna sequence dna of the required sudden change of encoding.

Can use the site-specific mutagenesis method to produce aminoacid deletion, insertion or replacement.Sequential amino acid deletion generally is about 1-30 residue, better is 1-10 residue, and generally is successive.Most preferred disappearance is those disappearances that can produce solubility ICAM-3.The best ICAM-3 that hydrophobic residue in diaphragm area or the ICAM-3 produces soluble form that strides by disappearance ICAM-3.

Aminoacid insertion be included in insert in the ICAM-3 encoding 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 (promptly 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-terminal of molecule, is beneficial to from recombinant host internal secretion 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 the feature of ICAM-3 molecule more accurately as expectation, but according to the form below replaces preferably.

The replacement that the original residue of table 1 is given an example

Can select has the still less replacement of conservative property than amino acid shown in the table 1, promptly select those for keeping (a) to replace polypeptide backbone structure in the zone, 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 immunological properties to molecule.In general, less conservative replacement be those wherein (a) glycine and/or proline(Pro) by another aminoacid replacement, or disappearance or be inserted into; (b) replace hydrophobic residue with hydrophilic residue; (c) replace (or being substituted) any other residue with halfcystine; (d) replace the residue that (or being substituted) has negative charge with residue with positive charge side chain; Or (e) replace the replacement that (or being substituted) has the residue of such side chain with the residue that has bulky side chain.

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

Be to improve the affinity of ICAM-3, can 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.Equally, can prepare this class lacks the CHO of N connection on IACM-1 or ICAM-2 listen with the source position mutant ICAM-3 molecule.

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

Then with the activity of suitable screening method according to the derivative of expection feature screening cellular lysate or purifying.For example, the change of the immunological characteristic of the available immunodetection of striving property unexpectedly detection functionality derivative is as the change to the affinity of known antibodies.Available method known to a person of ordinary skill in the art changes this proteinic character, as redox or thermostability, biology half life, hydrophobicity, to the susceptibility of proteolytic degradation effect, or with the carrier polymerization or aggregate into polymeric tendency etc.

The agonist of B.ICAM-3 and antagonist

" agonist " of ICAM-3 be meant and can improve or 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 combines with cell receptor.

" antagonist " of ICAM-3 is meant and can reduces or stop ICAM-3 to finish the compound of the ability of its any biological function.The example of such antagonist comprises ICAM-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, this paper reference is all classified in full in these patent applications of 446(as) described in the cell aggregation test can detect the LFA1 dependency and assemble, and therefore can be used for identifying the preparation that influences the ICAM-3/LFA-3 aggregation extent.Therefore, these detection methods can be used for identifying agonist and the antagonist of ICAM-3.Antagonist can play a role by recovering LFA-1 or ICAM-3 mediation accumulative ability.Whether in addition, available aforesaid method detects NIg (i.e. chemistry) preparation, serve as agonist or the antagonist of the ICAM-3/LFA-1 of mediation gathering to determine them.The periphery blood T cell that this cell aggregation detection method generally is to use to stimulate with PMA is finished.Therefore, can detect the cell aggregation of ICAM-3 mediation according to peripheral blood cells and combining of LFA-1.

C. anti-ICAM-3 antibody

Preferred immunoglobulins 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 the anti-ICAM-3 antibody that other are suitable for.

Anti-ICAM-3(or its functional deriv) antibody 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 peptide fragment can be introduced and make anti-ICAM-3 antibody in the suitable animal body.Immunized animal will produce polyclonal antibody in to its reaction process.Use the peptide fragment of ICAM-3 only can make with the peptide fragment that is used for immune animal in the regiospecificity antibody that reacts of contained epitope.

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

Moreover, can be by Selden, R.F.(European patent application publication No. 289,034) or people's (Science 236: 714-718(1987)) such as Selden R.F. method produce anti-ICAM-3 antibody.Specifically, available can The expressed ICAM-3 molecule or the cell of the suitable animal of its segmental carrier transfection.In the transfected cell of animal, produce ICAM-3 and will cause immune response in animal body, and then produce anti-ICAM-3 antibody by this animal.

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

Available several different methods is identified the hybridoma that can secrete the antibody that can combine with ICAM-3, to screen the hybridoma that obtains as stated above.In a preferred screening method, can suppress ICAM-3 expression, ICAM-1 and ICAM-2 non-expressing cell accumulative ability according to them and identify these molecules.Further then screening can suppress the antibody of this congregation, suppresses this congregation to determine whether they can combine by the member with 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 are by the antigen of antibodies.Might according to antibody with express LFA-1 but non-Table I CAM-3(or opposite) cell bonded ability, distinguish with CD-18 molecule family member bonded antibody and with ICAM-3 bonded antibody.The method that can use always by those of ordinary skills detects antibody with expression LFA-1 but do not express the ability that the cell of ICAM-3 combines.These methods comprise immunodetection (particularly using immunofluorescence), cell agglutination method, filter membrane combined techniques and antibody precipitation method etc.

In this preferred method, in conjunction with the cell of expressing ICAM-3, but debond is not expressed the ability of the cell of ICAM-3 and is selected antibody according to it.

Except the agonist and antagonist of above-mentioned ICAM-3, can be used according to the invention as the anti-antibody of anti-ICAM-3 antibody, can be in conjunction with other preparation for treating 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 (or repulsion) with ICAM-3 and combine.For example, can induce the anti-antibody of anti-ICAM-3 antibody, screening has the antibody in conjunction with the ability of the binding partner of natural ICAM-3 then, to obtain such antibody.

Because the molecule of CD-18 family can combine with ICAM-3, so use this molecule (as have the heterodimer of α and β subunit, or the molecule of only forming by α or β subunit or have molecule fragments single or two kinds of subunits) will combine the ICAM-3 that is present on the cell with cell competition.

Available several different methods is identified and titration anti-congregation antibody of the present invention.For example, can detect combination discriminatively and express the cell (as the T cell) of ICAM-3, and can not be in conjunction with the antibody of the cell of not expressing ICAM-3.The method (this paper reference is all classified in all these patent applications as) that is suitable for detecting cell aggregation has been described in the u.s. patent application serial number 07/045,963,07/115,798,07/155,993,07/189,815 or 07/250,446.In addition, the peptide fragment bonded ability of detectable antibody and 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 method to be provided, and wherein each method all can with ICAM-3 bonded antibody with distinguishing with CD-18 molecule family member bonded antibody discriminated union.

D. the preparation of preparation of the present invention

Can make preparation of the present invention by several different methods: natural method (produce the NIg antagonist of ICAM-3 as induced animal, plant, fungi, bacterium etc., or induced animal produce can with ICAM-3 bonded polyclonal antibody); 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 produce can with ICAM-3 bonded monoclonal antibody); Or recombinant technology (as using recombinant plasmid or virus vector generation preparation of the present invention in different host (as the mammalian cell of yeast, bacterium, fungi, cultivation etc.)).Select for use which kind of method will depend on factors such as whether convenient and productive rate size.But not necessarily only use above-mentioned a kind of method or technology to produce specific anti-inflammatory preparation; Can unite aforesaid method and the technology used for obtaining particular formulations.

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

Can use preparation of the present invention to regulate the various biological activity that mediates by ICAM-3.

A. inflammation-inhibiting

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

Term " inflammation " is meant the reaction of specificity system of defense, and the reaction of nonspecific defense system.

Term used herein " specificity system of defense " is meant the immune composition that the existence to specific antigens 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 antigen for example reaction, oneself immunological disease and the cell-mediated delayed type hypersensitivity (as seen in Mantaux test " positive " person) of T of rubella virus.Other examples of the Inflammatory response of specificity system of defense comprise that chronic inflammation disease reaches the rejection to entity transplanted tissue and organ (bribing transplanting as kidney and bone).

The reaction of this paper said " nonspecific defense system " is meant the reaction by the white corpuscle mediation that can not produce immunological memory.This class cell comprises granulocyte and scavenger cell.As described herein, if inflammation is reaction by the nonspecific defense system to be caused or mediate, or get in touch with the reacting phase of nonspecific defense system, then this inflammation says that promptly becoming is the result of non-specific system of defense reaction.To small part be because the example of the inflammation that the reaction of nonspecific defense system causes comprises the inflammation relevant with following pathological conditions: adult respiratory distress syndrome (ARDS) or septicemia, wound or posthemorrhagic multiple organ injury's syndromes, the reperfusion injury of cardiac muscle or its hetero-organization, acute glomerulonephritis, reactive arthritis, have the tetter that acute inflammation changes, acute purulent meningitis or other inflammatory disease of central nervous system such as apoplexy, thermal damage, hemodialysis, the white corpuscle deformity, ulcerative colitis, Crohn disease, necrotizing enterocolitis, granulocyte transfers relevant syndromes and cytokine inductive toxicity.

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

Anti-CD-18 molecule family member's monoclonal antibody can suppress leukocytic many adhesion dependency functions, comprise the (Haskard that combines with endotheliocyte, D.et al., J.Immunol.137: 2901-2906(1986)), homotype adheres to (Rothlein, R.et al., J.Exp.Med.163: 1132-1149(1986)), antigen and mitogen inductive lymphopoiesis (Davignon, 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 functions, as cellular toxicity T cell (Krensky, A.M.et al., J.Immunol.132: 2180-2182(1984)), scavenger cell (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 white corpuscle to the adherent ability of suitable cell matrix, and the latter suppresses the biological function be associated with above-mentioned combination again 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 can with ICAM-3 bonded monoclonal antibody as mammiferous anti-inflammatory preparation.This class preparation and common anti-inflammatory agent obvious different be they can optionally suppress adhesive attraction, do not have conventional dose common other pairs effect (as nephrotoxicity).

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

1. the inhibitor of delayed type hypersensitivity

ICAM-3 partly mediates to forming inflammatory reaction such as the necessary adhesion process of delayed hypersensitivity.Therefore, can with ICAM-3 bonded antibody weaken 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 particularly its soluble form of ICAM-3() or its functional derivatives suppress delayed hypersensitivity.

Can develop these potential treatments by dual mode uses.The first, can there be the patient of delayed hypersensitivity 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 antigenic patients 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 response.Therefore, additional antigen in anti-ICAM-3 antibody, the antigen that the temporary transient tolerance of patient will be contacted once more.

2. treatment chronic inflammatory disease

Patient LAD in default of LFA-1 can not produce Inflammatory response, also will suppress Inflammatory response so can believe the antagonistic action of the native ligand of LFA-1.The ability of anti-ICAM-3 antibody inflammation-inhibiting 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, Reynolds syndrome, rheumatoid arthritis and is provided the foundation.Also can use these Antybody therapy psoriasiss.

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 of treatment that the anti-inflammatory agent of inflammation-inhibiting q.s is provided, in order to suppress this inflammatory and immune response (promptly stop or weaken it).The anti-inflammatory agent that is suitable for comprises: can with ICAM-3 bonded antibody, said antibody can with ICAM-3 bonded fragment, 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 basically.Particularly preferably be the anti-inflammatory agent of forming by the soluble functional derivative of ICAM-3.Such anti-inflammatory treatment can comprise to patient and uses the additional composition that is selected from down series preparation that has: can with the NIg antagonist of LFA-1 bonded antibody, 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 response 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 littler (promptly 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 the immune antagonist that is suitable for comprises but not only is confined to dexamethasone, azathioprine, ICAM-1, ICAM-2 or cyclosporin A.

3. treatment nonspecific inflammation

Many Inflammatory responses are because the reaction of " nonspecific defense system " causes, and are by the cell mediated that can not produce immunological memory.This class cell comprises granulocyte and scavenger cell.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, we can say that promptly this inflammation is the result of non-specific system of defense reaction.Comprise the inflammation of closing by the example of the inflammation due to the reaction of nonspecific defense system at least in part: adult respiratory distress syndrome (ARDS) or be secondary to septicemia with following pathological conditions sample, wound, or hemorrhage multiple organ injury's syndromes, the reperfusion injury of cardiac muscle or its hetero-organization, acute glomerulonephritis, reactive arthritis, with acutely inflamed tetter, other inflammatory diseasess such as the apoplexy of acute purulent meningitis or central nervous system, thermal damage, hemodialysis, the white corpuscle deformity, ulcerative colitis, Crohn disease, necrotizing enterocolitis, granulocyte transfers syndromes and cytokine inductive toxicity.

Anti-inflammatory agent of the present invention be can the antagonism granulocyte on CD-18 mixture and the interactional compound of endotheliocyte.This class antagonist comprises: the NIg antagonist of the functional derivatives of ICAM-3, ICAM-3, the ICAM-3 except that ICAM-1, ICAM-2 or CD-18 molecule family member.

B. the inhibitor of organ and tissue rejection

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 cause by any cell adhesion dependency 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 Mammals or tissue (as kidney) or unsubstantiality organ or tissue (as marrow), perhaps improve oneself immune response.Importantly use the monoclonal antibody that to discern ICAM-3, so as to can between the individuality of HLA mismatch, carrying out organ transplantation.

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 diagnostic reagent such as Sigma I8405, Interferon, rabbit, tissue type plasminogen activator or mouse monoclonal antibody can damage the 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 diagnostic reagent to unite use.Add antibody and can prevent the identification of recipient, thereby prevent that the recipient from producing the immune response of anti-these medicaments above-mentioned medicament.Eliminate the treatment or the diagnostic reagent that can make patient accept to add after the above-mentioned immune response.

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

D. metastases 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 that the preparation (if can with ICAM-3 bonded antibody, the toxin derived products of said antibody, said antibody can be in conjunction with the fragment of ICAM-3, said segmental toxin derived products, the functional deriv of pure ICAM-3, ICAM-3 or the NIg antagonist of the ICAM-3 except that ICAM-1 or ICAM-2 basically) of the amount that can suppress said transfer completely is provided.

In another example of the present invention, provide the method for the growth of tumour cell that suppresses expression ICAM-3.Specifically, this method comprises that patient to this treatment of needs provides the therapeutical agent of q.s.The therapeutical agent that is suitable for comprise can with ICAM-3 bonded antibody, the toxin derivant of said antibody, said antibody can with the toxin derived products of ICAM-3 bonded fragment, said segmental toxin derivant, CD-18 molecule family member's toxin derived products or CD-18 molecule family member's functional deriv.

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

E. suppress the cellular invasion that HIV infects and prevent the HIV infection

In another embodiment of the present invention, the method that provides a kind of HIV of inhibition to infect, this method comprises to being subjected to HIV the infected to use the HIV infection inhibitor of significant quantity.Suppress the method that HIV-1 infects though the present invention relates to particularly, it should be noted that this method also is applicable to the HIV varient (as HIV-2) of the cells infected in some way that any available preparation of the present invention suppresses.With regard to purpose of the present invention, these varients 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 the LFA-1 binding partner in some cases, thereby promoted cell-cell adhesion reaction, be increased duration of contact of infected cell and non-infected cells, help 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 particularly infection that is 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 cell expressing 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 cell expressing 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.Under conventional administration situation,, we can say that promptly dosage is enough to " inhibition " HIV and infects if institute is enough to weaken to dosage or stop HIV to infect.These preparations are provided for contacted HIV or are subjected to the patient that HIV infects.

In handling the HIV infection, preparation of the present invention can be used for " prevention " or " treatment " purpose.When being used to prevent, can be before any virus infection 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 used to stop or weaken continue after HIV infect.Be when detecting the cell that is infected by the virus, (or in not long-time afterwards) to provide said preparation when being used for the treatment of.The further infection that the treatment administration is intended to alleviate HIV.

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

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

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

(II) can with ICAM-3 bonded antibody, and

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

(IV) can with CD4 bonded molecule, better be antibody or antibody fragment.

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

Anti-migration agent of the present invention comprises the fragment of ICAM-3, the ICAM-3 that can damage the ability that T cell that HIV infects combines with the LFA-1 part, functional deriv or the anti-ICAM-3 antibody of ICAM-3.Can in conjunction with the antibody of ICAM-3 will be by infringement HIV infections the cell bonded ability of ICAM-3 and expression CD11/CD18 acceptor of T cell expressing suppress cell migration.In order to destroy the ability of cell and CD11a/CD18 receptors bind, might use can be in conjunction with the antibody of ICAM-3.

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

Can use these compounds for " prevention " or " treatment " purpose.When being used to prevent, 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 used to stop or weaken continue after by the migration of the cell of virus infection.When being used for the treatment of, can when detecting the T cell of virus infection, (or in not long-time afterwards) provide said preparation.The treatment administration is to be used to weaken the further ability of migration of these T cells.

Therefore, preparation of the present invention can be used on virus infection 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 infection after.

F. treat asthma

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

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 the anti-ICAM-3 antibody of ICAM-3.In conjunction with the antibody of ICAM-3 by destroying the migration that ICAM-3 that expresses on these cells and the cell bonded ability of expressing the CD11/CD18 acceptor suppress eosinophil.

The consumption of anti-asthmatic agent of the present invention should be enough to reduce or slow down the seriousness and the time length of symptoms of asthma.

Preparation of the present invention can use separately, also can with one or more other anti-asthmatic agents (as methyl xanthine, theophylline), beta-adrenergic agonist (as catecholamine, resorcin, saligenol, ephedrine), glucocorticosteroid (as hydrocortisone), chromone (as Sodium Cromoglicate) 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 to prevent, can before occurring, symptoms of asthma use.The purpose of said preparation prevention administration is intended to prevent or alleviate the symptoms of asthma of secondary.When being used for the treatment of, must (or thereafter soon) medication when symptoms of asthma 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 severity or the shortening duration of seizure of alleviating outbreak) before asthma attack occurring, perhaps use after outbreak.

G. diagnosis and prognosis are used

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

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

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

For example, by anti-ICAM-3 antibody of radioactivity mark or antibody fragment, might detect antigen with radioimmunology.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), visible particularly Chard, the exercise question that T. writes is " An Introduction to Radioimmune Assay and Related Techniques " chapter (this article is classified this specification sheets reference as).In addition, also available fluorescent chemicals, enzyme or other suitable markers are pressed the currently known methods traget antibody.

Except determining inflammation part, also can use and can and utilize the substratum commonly used that is suitable for doing body fluid analysis with ICAM-3 bonded antibody, whether there is round-robin ICAM-3 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 in the amniotic fluid with the complication of gestation time.Can carry out this detection to any biological fluid, but said biological 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 of treatment of ICAM-3 to patient.Wherein useful especially is the therapeutic activity peptide fragment of the ICAM-3 of soluble form.

Available synthetic method, DNA recombinant technology, albumen hydrolysis or share aforesaid method and make ICAM-3 and functional deriv thereof.Can use the ICAM-3 functional deriv that has additional amino-acid residue (in order to the activity of raising), to improve the result of treatment of ICAM-3 with carrier link coupled ability or enhancing 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 the biology or the pharmacological activity that maybe can influence ICAM-3.

Basically do not have naturally occurring material if contain in the preparation of ICAM-3 molecule of antibody of the present invention and this paper discussion, we can say that promptly these antibody and ICAM-3 molecule are " not having natural pollutant basically ".

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

ICAM-3 or by the deutero-molecule can use separately, also can share with ICAM-1 and/or ICAM-2.Anti-ICAM-3 antibody or can with ICAM-3 or by other molecules of deutero-molecule bonded also can use separately or share with anti-ICAM-1 antibody and/or anti-ICAM-2 antibody.When using antibody to patient or can be with ICAM-3 bonded antibody fragment the time, maybe when use ICAM-3(or its fragment, variant or derivative to patient) time, dosage can be different according to factor such as year the making of patient, body weight, height, sex, general curative situation, medical history.Generally speaking, the dosage of antibody is the 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 the 1pg-10mg/Kg(patient body weight more fortunately) in the 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 the antibody combined use of anti-ICAM-2, can reduce the effective dose in the treatment.The notion used as text, when the administration time of two kinds of compounds very near so that can in the patients serum, detect these two kinds of compounds simultaneously the time, promptly we can say a kind of compound and second kind of compound Combined Preparation.

Can be with the route of administration of ICAM-3 bonded antibody and ICAM-3 itself in intravenously, intramuscular, subcutaneous, the intestines, part or the outer approach of gi tract.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 promptly be said to be on the physiology effectively.For example, when using preparation inflammation-inhibiting of the present invention to patient, can " suppress " inflammation completely as dosage, then said preparation be exactly on the physiology effectively.

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

Therefore, anti-inflammatory agent of the present invention can use (be used to suppress may occur inflammation) or in inflammation the back use take place before inflammation takes place.

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

Can be according to the known method of 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.Carrier that is suitable for and prescription thereof (preparation that comprises other people protein such as human serum albumin etc.) are at Remington ' s Pharmaceutical Sciences(16th ed., Osol, A., Ed., Mack, Easton PA(1980)) be described in.In order to make the pharmaceutically acceptable composition that is suitable for clinical administration, these compositions can contain the carrier of the preparation of the present invention of significant quantity together with appropriate amount.In addition, can make antibody humanization of the present invention, be easier to be accepted by human body thereby become by chimeric or CDR grafting.In No. 9009548.0,9009549.8, UK Patent Application and PCT application PCT/US91/02942 number 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 the method for pharmacy control useful effect phase in addition.Can control the release of these preparations by using polymkeric substance compound or absorption preparation of the present invention.Can controlled travelling speed and cycle be adjusted to a specific degree by the concentration of selecting suitable macromole matrix and macromole that change adds.Discharging the another kind of possible method of control action kou time by the control preparation is that preparation of the present invention is incorporated in the particle of polymer materials such as polyester, polyamino acid, aqueous gel, poly(lactic acid) or ethylene vinyl acetate copolymer.In addition, can also use condensation technique or interface polymerization, utilize gelatin or poly-(different methyl acrylate) to be embedded in these materials in the microcapsule or be embedded in colloid medicament transport system for example in liposome, albumin microsphere, microemulsion, molecule and tiny capsules or the macromole emulsion.

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

(1) anti-inflammatory agent (as can with ICAM-3 bonded antibody, can with the fragment of the said antibody of ICAM-3 bonded, the functional deriv of ICAM-3, ICAM-3 or the NIg antagonist of the ICAM-3 except that ICAM-1 and ICAM-2), and (b) at least a immune antagonist.The example of the immune antagonist that is suitable for comprises: dexamethasone, azathioprine and cyclosporin A.

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

Embodiment 1

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

By preceding method clone and lymphocyte are adhered on the plate of LFA-1 bag quilt (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 m ²Loading 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 site ₂/ 0.5%HSA(detects substratum) wash each hole.The anti-LFA-1 α of TS1/22(that adds 1/200 dilution in microtiter well) and under room temperature be incubated 30 minutes, 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 phytohemagglutinin (PHA) culturing cell with generation PHA parent cell.With fluorochrome BCECF(Molecular Probes Inc.) labeled cell, wash behind the cell resuspending in detecting substratum.Be incubated 45 minutes 4 ℃ of ascites, carry out the MAb pre-treatment, in every hole, add 10 then with pair cell with cell and 1/200 dilution ⁵Individual cell.37 ℃ of insulations 1 hour make cell adhesion 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,, from flat board, remove lymphocyte with 100 ψ attack substratum 8 times.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 fluorescence concentration analyzer (Baxte) direct quantitative is analyzed the fluorescence volume in the 96 hole flat boards.All used MAb are saturation concentration (ascites of 1/200 times of dilution), and comprise the anti-CD11a of TS1/22(, IgCl) (Sanchen-Madrid et al., Proc.Natl.Acad.Sci.USA.75: 7489-7493(1982)), the anti-ICAM-1 of RR 1/1(, IgGl) (Rothlein et al., J.Immunol.137: 1270-1274(1986)), the anti-ICAM-2 of CBR-1C 2/2(, IgG2a) (de Fougerolles et al., J.Exp.Med.Submitted(1991) (wait to publish)), and the anti-ICAM-3 of CBR-IC3/1(, IgGl).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 filter out 600 myelomatosis, thereby make CBR-IC3-1 according to its LFA-1 bonded ability that suppresses SKW3 and purifying.CRB-IC3/1 performance not with the LFA-1 reaction of purifying, also not with COS cell response (data not shown goes out) with ICAM-1, ICAM-2 or LFA-1cDNA transfection.Shown the result of one of four representative experiments, wherein the error strip chart shows a standard deviation.

The embodiment II

The wandering cells 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.By the dextran stated precipitation and Ficoll-Hypaque(1.077) centrifuging makes peripheral blood lymphocytes (PBMC).By the red corpuscle that reclaims neutrophil and pollute in the cell precipitation group through hypotonic dissolution destruction.Utilize and to carry out the cell counting analysis with vertical light scatter forward, so that monocyte is separated with the T cell, and confirm it with monocyte and T cell-specific MAb.With the plastics adhesion method from PBMC lymphocyte-rich and in the presence of 10 μ g/ml phytohemagglutinin (PHA) culturing cell.With EPICSV wandering cells counter analytic sample, and use EPICS Immune-Brite fluorescent bead (Coulter) that fluorescence is carried out quantitatively with the calibration cell counter.On the resting cell expression ratio CD3 of ICAM-3 or the big 2-3 of LFA-1 doubly, and the ICAM-3 of monocytes manys 3-4 doubly than LFA-1.The expression level of the last ICAM-3 of neutrophil(e) cell and Mac-1(CD11b/CD18) equate.Handling cell with Phospholipase C shows and does not exist and PI bonded ICAM-3.

The embodiment III

The immunoprecipitation of ICAM-3

By state method (Kishimoto et al., J.Biol.Chem.264: 3588-3595(1989)) with iodine ( ¹²⁵I) pair cell carries out surface markers.Use Triton X-100(1%) dissolved cell.Add ox IgG link coupled Sepharose predefecation lysate, then with suitable MAb bonded Sepharose insulation 2 hours.Wash the sample globule and in the sample buffer that contains 50mM Tris, 1%SDS and 1% 2 mercapto ethanol, boil.In the damping fluid that does not contain 2 mercapto ethanol, boil non-reducing sample, and handle with 20mM sulphur ethanamide.By stating method (Nature 227: 680-685(1970) for Laemmli, U.K.) analytic sample on 7% vertical panel polyacrylamide gel, and manifest protein with the radioautograph method.By former described method (Tarentino et al., Biochemistry 24: 4665-4671(1985)), use certain density N-glycanase (Genzyme) to handle 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 600,000,6 N connection site of ICAM-2(molecular weight, backbone molecule amount 28,383) then be high glycosylation.

* described in material and method, pass through the film expression that the immunofluorescence flow cytometry is determined.Resulting value is at least twice result of experiment.Calibrate hematimeter with fluorescent bead, so that 1 unit is approximately 10 ³The fluorescein equivalent (Coulte Diagnositics, Hialeah, FL).

* mixes clone and comprises: Human umbilical vein endothelial cells, 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.

Claims (43)

1, the ICAM-3 or its functional deriv that do not contain natural pollutant substantially.

2, can the encode recombinant DNA molecules of ICAM-3 or its functional deriv.

3, according to the recombinant DNA molecules of claim 2, wherein said molecule can also be expressed ICAM-3 or its functional deriv in cell.

4, can with the molecule bonded antibody or the antibody fragment that are selected from ICAM-3 and its functional deriv.

5, according to the antibody of claim 4, wherein said antibody damages said molecule and acceptor molecule bonded ability with combining of said molecule.

6, according to the antibody of claim 5, wherein said acceptor is LFA-1.

7, according to the antibody of claim 5, wherein said acceptor is Mac-1.

8, according to the antibody of claim 5, wherein said acceptor is p150,95.

9, can produce the hybridoma of the monoclonal antibody of claim 4.

10, produce the method for required hybridoma, said cell produce can with ICAM-3 or its function fragment bonded antibody, this method comprises the following steps:

(a) the ICAM-3 immune animal of usefulness purifying,

(b) splenocyte of this animal and myeloma cell line are merged,

(c) make the spleen of fusion and the hybridoma that the myeloma cell forms secretory antibody, and

(d) ability that produces the antibody that can combine with ICAM-3 according to required hybridoma is screened this hybridoma.

11, regulate the method for the cell biological function of ICAM-3 mediation, wherein said method comprises that the patient to this treatment of needs provides the ICAM-3 conditioning agent of significant quantity, said ICAM-3 conditioning agent be selected from can with ICAM-3 bonded antibody, said antibody can with the NIg antagonist of ICAM-3 bonded fragment, ICAM-3 and functional deriv thereof and the ICAM-3 except that ICAM-1, ICAM-2 or CD-18 molecule family member.

12, according to the method for claim 11, the biological function of wherein said ICAM-3 mediation is Inflammatory response, and said ICAM-3 conditioning agent is to offer said patient's with the amount that can suppress said inflammation completely.

13, according to the method for claim 12, wherein said Inflammatory response is to being selected from the specificity inflammation that occurs in following one group of state response: delayed hypersensitivity, psoriasic symptom, oneself immunological disease, organ transplantation or tissue grafts repulsive interaction.

14, according to the method for claim 13, wherein said organ transplantation is that the parenchymatous organ transplants.

15, according to the method for claim 14, wherein said organ transplantation is renal transplantation.

16, according to the method for claim 13, wherein said organ transplantation right and wrong parenchymatous organ transplants.

17, according to the method for claim 16, wherein said organ transplantation is a bone marrow transplantation.

18, according to the method for claim 13, wherein said oneself immunological disease is selected from Reynolds syndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis and lupus erythematosus.

19, method according to claim 13, wherein said inflammatory reaction is the specificity inflammation that forms in to one group pathological state reaction below being selected from: adult respiratory distress syndrome (ARDS), be secondary to septicemia, wound or hemorrhage multiple organ injury's syndromes, the reperfusion injury of cardiac muscle or its hetero-organization, acute glomerulonephritis, reactive arthritis, with acutely inflamed tetter, acute suppurative arthritis or other inflammatory disease of central nervous system such as apoplexy, thermal damage, hemodialysis, the special-shaped disease of white corpuscle, ulcerative colitis, Crohn disease, necrotizing enterocolitis, granulocyte transfer the toxicity that relevant syndromes and cytokine are brought out.

20, according to the method for claim 11, the biological function of wherein said TCAM-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.

21, according to the method for claim 11, the biological function of wherein said ICAM-3 mediation is the leukocytic blood vessel external migration of virus infection, 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.

22, according to the method for claim 11, wherein the cell of said virus infection is infected by HIV.

23, according to the method for claim 11, wherein said ICAM-3 biological function is the migration of the cell relevant with asthma reaction, and said ICAM-3 conditioning agent is to offer said patient's with the amount that can suppress the cell migration relevant with asthma completely.

24, suppress the method that white corpuscle is infected by HIV, wherein said method comprises the HIV-1 infection inhibitor that uses significant quantity to patient contacted or that be subjected to HIV impression, said preparation be selected from can with ICAM-3 bonded antibody, the toxin derived products of said antibody, said antibody can with the functional deriv of ICAM-3 bonded fragment, said segmental toxin derived products, ICAM-3; The NIg antagonist of ICAM-3 except that the member of ICAM-1, ICAM-2 or CD-18 molecule family, and the member's of CD-18 molecule family toxin deutero-functional derivatives.

25, according to the method for claim 24, wherein said HIV is HIV-1.

26, the method that suppresses the growth of tumour cell of expression ICAM-3, this method comprises that the patient to this treatment of needs provides the toxin that can suppress said increment completely, said toxin be selected from can with ICAM-3 bonded antibody, the toxin derived products of said antibody, said antibody can with ICAM-3 bonded fragment, said segmental toxin derived products, CD-18 molecule family member's toxin derived products, and the member's of CD-18 molecule family toxin deutero-functional derivatives.

27, the method that suppresses the growth of tumour cell of expression LFA-1, said method comprises that the patient to this treatment of needs provides the toxin that is enough to suppress said increment, and said toxin is selected from the functional derivatives of toxin deutero-ICAM-3 and toxin deutero-ICAM-3.

28, according to each method among the claim 11-27, wherein said method also comprises uses one or more preparations be selected from following one group: can with LFA-1 bonded antibody, said antibody can with LFA-1 bonded fragment, the NIg antagonist of LFA-1, can with ICAM-1 bonded antibody, said antibody can with ICAM-1 bonded fragment, can with ICAM-2 bonded antibody, said antibody can with ICAM-2 bonded fragment.

29, according to each method among the claim 11-27, wherein said ICAM-3 conditioning agent is administration in enteron aisle, outside the gi tract, in local, suction or the nose.

30, according to each method among the claim 11-27, wherein said ICAM-3 conditioning agent is used to prevent purpose.

31, according to each method among the claim 11-27, wherein said ICAM-3 conditioning agent is used for the treatment of purpose.

32, according to the method for claim 29, wherein said gi tract external administration approach is intramuscular, intravenously or subcutaneous administration.

33, contain the medical composition that is selected from following one group preparation: can with ICAM-3 bonded antibody, said antibody can with ICAM-3 bonded fragment, ICAM-3, the functional deriv of ICAM-3, and the NIg antagonist of the ICAM-3 except that the member of ICAM-1, ICAM-2 or CD-18 family.

34, according to the medical composition of claim 33, it also contains immunosuppressor in addition.

35, contain and pharmaceutically acceptable carrier-bound medical composition according to each ICAM-3 conditioning agent in the claim 11,24,26,27 or 28.

36,, wherein be combined with at least a other immunosuppressor according to the medical composition of claim 35.

37, contain the antibody molecule of with good grounds claim 4 or it is segmental, and be combined with the therapeutic composition of pharmaceutically acceptable thinner, vehicle or carrier.

38, contain antibody molecule or its segmental diagnosis composition that is the mark pattern that can detect according to claim 4.

39, detect the method that whether has the cell of expressing ICAM-3, this method comprises:

(a) extract of said cell of insulation or said cell in the presence of a kind of nucleic acid molecule, said nucleic acid molecule can be hybridized with ICAM-3mRNA; And

(b) determine whether said nucleic acid molecule hybridizes with the complementary nucleic acid molecule that is present in said cell or the said cell extract.

40, the method for the tumour cell of diagnosis Mammals expression in vivo ICAM-3, this method comprises:

(a) use for said diagnosis object to contain the traget antibody that can detect or its can combine its segmental composition with ICAM-3, and

(b) detect and the said antibody of said ICAM-3 bonded.

41, the method for the inflammation in the diagnosis mammalian body, this method comprises:

(a) tissue sample with said diagnosis object can be incubated with antibody or its segmental composition that the cell of expressing ICAM-3 combines with containing, and

(b) detect and the said antibody of said cell bonded.

42, whether have the method for ICAM-3 in the diagnosis mammalian body fluid sample, this method comprises:

(a) body fluid example with said diagnosis object is incubated with containing antibody or its segmental composition that can combine with ICAM-3, and

(b) detect and said body fluid bonded antibody.

43, according to the method for claim 42, wherein said body fluid is selected from blood, serum, blood plasma, synovia, amniotic fluid, cerebrospinal fluid or urine.

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