FI120496B - Human endothelial cell molecule affecting lymphocyte binding - Google Patents

Description

Human sndoteeil cell molecule that acts on lymphocyte binding

FIELD OF THE INVENTION

This invention relates to a novel human endothelial cell adhesion antigen-5 gene, called VAP-1, and to a method for producing a monoclonal antibody, 1B2, which recognizes VAP-1 antigen,

BACKGROUND OF THE INVENTION

Most mature lymphocytes circulate continuously between blood and lymphoid organs (Butcher, E, C. Curr. Top. Microbiol. Immunol. 128, 85-10 (1986)). The lymphocytes are cleared from the blood by recognition and adherence to the endothelial cells of the blood vessels. The migration of lymphocytes enables all lymphocytic-nonspecific immune reactions in every part of the body, and facilitates the intracellular interactions required for the generation and control of the immune response. Adherence of lymphocytes to endothelial cells is affected by the interaction of complementary adhesion molecules expressed in both cell types (Springer, TA, Nature 346, 425 (1990); Stoolman, LM .. Cell 56, 907 (1989); Osborn, L, Cell 62, 3 (1990); Pober and Cotran, Transplantation 50, 537 (1990); Butcher, EC, Cell 67, 1033 (1991)). Under normal conditions, the lymphocytes bind mainly to different types of convex postcapillary veins called High Endothelial Venule (HEV). Functionally distinct lymphocyte-HEV recognition systems that contribute to the transport of lymphocytes to peripheral lymph nodes, intestinal lymphoid tissue, conjunctiva and skin by organ specificity have been described in the literature 25 (Butcher, EC. Jalkanen, S., et al., Science 233, 556 (1986); Picker, LJ, et al., Nature 349, 796 (1991)). In the inflammatory situation, activation of the endothelial cell causes changes in the state of the adhesion molecules, which greatly affects the number and type of leukocytes that migrate into the inflamed tissue. Thus, endothelial cells play a key role in the control of the local immune response, and a detailed understanding of the mechanism by which lymphocytes circulate and exit the bloodstream may, of course, provide new opportunities for clinical modulation of the inflammatory response.

2

Since the endothelial cell ligands affecting tissue-selective migration of human lymphocytes are largely unknown, there is a great need to identify such molecules.

In June 1991, the present inventors presented preliminary results in a conference abstract of the European Federation of Immuno-Nologicai Socieies, which suggested the existence of an antigen known to previously unknown endothelial cell that affects lymphocyte HEV binding. (Salmi et al., EFIS 11th Meeting Abstracts, 21-12; 1991).

SUMMARY OF THE INVENTION

Recognizing the importance of inflammation control and the need for an understanding of tissue-selective endothelialized figs affecting lymphocyte transport, the present inventors seek to identify molecules expressed in human articular veins. These studies culminate in the identification of a new human endothelial cell molecule that is involved in lymphocyte binding, is VAP-1, and in the production of monoclonal antibodies that recognize this molecule. These antibodies are useful in assays for the quantitative and qualitative determination of VAP-1 levels, as well as in a clinical treatment setting that reverses the function of the VAP-1 molecule in a patient in need of treatment.

Thus, the present invention primarily relates to VAP-1 protein, which is substantially free of the natural contaminants associated with such VAP-1 protein in a human or animal.

The invention further relates to isolated VAP-1 protein.

The invention further relates to methods of making antibodies, in particular monoclonal antibodies, to VAP-1 -25 protein, and to compositions containing such antibodies. The present invention describes a monoclonal antibody, 1B2, as well as a hybrid masoulin (DSM ACC2041) producing it.

The invention further relates to a method of counteracting VAP-1-mediated lymphocyte binding to endothelial cells, which method inhibits the VAP-1-mediated adipose reaction of lymphocytes and endothelial cells by administering a VAP-1 binding compound in an amount sufficient for Blok-endothelial VAP-1 , especially in cases where the ad-35 hessian response of lymphocytes and endothelial cells is associated with a disease such as inflammation (chronic or acute) arthritis, rheumatism, dermis, inflammatory bowel disease and autoimmune disease.

i j 3

BRIEF DESCRIPTION OF THE IMAGES

Figure 1. Occurrence of VAP-1 in human tissues. (A) In inflamed synovial membrane, monoclonal antibody 1B2 strongly stains HEV-like vessels. (B) In VTE, VAP-1 expression in HEV ranges from can-5 larvae (arrow) to weak (arrow) or negative. (C) Immuno-fluorescence staining of Nielursa demonstrates significant expression of VAP-1 on the luminal surface of the vessel (arrows). (D) Only a few poorly discolored HEVs (arrows) are seen in the appendix. Magnifications: A 100x, B 250x, C and D 400x.

Figure 2. VAP-1 is a 90 kD protein. Lane 1: Immuno-10 silver staining of purified VAP-1, Lanes 2-3: 125i-labeled laryngeal splenic tissue cells were immunoprecipitated with either antibody 1B2 (lane 2) or control antibody 3G6 (lane 3). 180-200 kD bands don't always perform. Molecular weight standards Left lymphocyte-free nerve extracts were solubilized in lysis buffer (150 mM NaCl, 10 mM Tris-base, 0.15 mM MgCl 4, 1% NP-40, 15 µm PMSF and 1% aprotinin) overnight at 4 ° C. The lysate was centrifuged at 10,000 g for 30 min at 4 ° C. The lysate was pre-purified on a Sepharose CL-4B (Pharmacia, Sweden) column. The lysate was then passed through three CNBr-activated Sepharose columns (Sepharose-4B (Pharmacia) derivatized with either normal mouse serum, irrelevant IgGi antibody or 1B2 antibody (5 20 mg / ml, 5 ml column). ) The columns were washed thoroughly with lysis buffer, and the material bound to the 1B2 column was then eluted using 50 mM triethanothiamine, matured, dissolved in SDS-PAGE (7.5%, reduced) and visualized by silver staining.

Figure 3. VAP-1 is involved in lymphocyte binding to HEV. Binding of lymphocytes to the umbilical cord, peripheral femoral muscle (PLN), synovial membrane, and HEV of granulocytes was determined, as well as binding of granulocytes to the tonsil HEV in the presence or absence of 1 B2 antibody, using in vitro binding. The results of three independent experiments with standard errors are presented as percentages relative to control binding (100% = 30 cells bound to 3G6-treated sections).

Figure 4. Isolated VAP-1 supports lymphocyte binding, immuno-purified VAP-1, and control proteins (1E12; another endothelial elution molecule that supports lymphocyte binding, and BSA) were absorbed on glass, and lymphocyte binding was determined. (A) Results of two independent experiments, expressed as a percentage of control binding (100% = number of cells bound to plate-bound VAP-1 or 1E12 after treatment with 3G6). Non-specific binding (BSA to protein binding) is subtracted from all assays. (B) Binding of lymphocytes to VÄP-1 coated wells in the presence of 3G6 antibody. (C) Binding of lymphocytes to VAP-1 coated wells in the presence of 1 B2 antibody.

The VAP-1 and 1E12 proteins were affinity purified from the tonsil extracts as shown in Figure 2. Purified VAP-1, 11 E12 and heat-inactivated BSA were diluted in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM MgCl 2, 2 mM Ca-Cl 2 using 0.01% β-octyl glucopyranoside as a detergent. Proteins were fixed in asiatic wells (Lab-Tek chamber plate, NUNC) for 16 h at + 4 ° C. Blocking was performed for 30 min at room temperature with PBS supplemented with 1 µg / ml BSA, after which 1B2 and 3G6 supernatants were added to the wells and incubated for 30 min at room temperature. Simultaneously, freshly isolated peripheral blood mononuclear cells were incubated in RPMI 1640 containing 10% FCS and 10 mM Hepes for 1 hour at 37 ° C to remove bottom-adherent monocytes. Non-adherent lymphocytes (1.8x10® cells / well) were added to each well in 100 µl RPMI 1640. Incubated for 30 min at 37 ° C, with non-adherent cells removed. The well partitions were removed, the plates were washed in weak PBS current and fixed in cold PBS containing 1% glutaraldehyde. The cells were then stained with Diff-Quick staining medium.

The adherent cells were quantitated by visually determining the number of cells in each well (total surface area 50 mm 2 / sample).

Figure 5. VAP-1 is increased in inflamed bowel. When normalizing, only a few weakly positive veins are present in Lamina prapria (A, at this point the veins are virtually VAP-1 negative). Multiple VAP-1-positive vessels (arrows) are seen in the inflamed vessel (ulcerative colitis), both in B) Lamina propria and C) in organized lymphoid collections, Endogenous peroxidase-containing cells (mast cells) show non-specific reactivity, e. epithelial cells; ip, Lamina propria. Magnification 25Öx.

Figure 6, Induction of VÄP-1 in chronic dermatosis. Skin biopsies from normal area (A) and psoriasis lesion (B) of same patient | that VAP-1 is induced in dermal sites of inflammation (arrows). Peri-vascular leukocyte infection is seen around VAP-1 positive vessels. e, the epidermis. Magnification 200x. C) VAP-1 expression (estimate + -> ++++) in 35 normal and diseased skin was determined from parallel biopsies (normal and lesion) from the same patient. The numbers in parentheses represent the number of patients in each group.

Figure 7. Inflammation-induced VAP-1 affects lymphocyte binding. Icicles were performed on a binding assay to examine peripheral 5 lymphocyte binding in Factor VIII positive veins in inflamed Lamina propria. The inhibition test was performed by preincubating tissue sections with antibodies 1B2 and 3G6. Results are expressed as percent control control with standard error (i.e., PBL binding in the presence of 3G6 is defined as 100%).

DETAILED DESCRIPTION OF THE INVENTION

Interaction between leukocyte surface receptors and their ligands in vascular endothelial cells is critical for the movement of lymphocytes between blood and various lymphatic tissues and for the transport of leukocytes from the veins to the sites of inflammation. We describe herein a novel 15 90 kD human endothelial cell adhesion molecule (VAP-1) as defined by monoclonal antibody 1B2. VAP-1 is predominantly expressed in HEVs of the synovial membrane, peripheral lymph nodes and tonsil, and 1B2 significantly inhibits lymphocyte binding to HEV in these tissues, unlike intestinal lymphoid tissue. In addition, lymphocytes bind to immunoaffinity-secreted VAP-1 in a manner that is inhibited by 1B2 antibodies. Occurrence, molecular weight and functional properties indicate that VAP-1 is a novel endothelial ligand for lymphocytes. We conclude that VAP-1 is a novel vascular molecule directly involved in the transport of lymphocytes in man. Monoclonal antibodies were generated by preliminary immunization of a VAP-1 protein by immunization with a suitable animal, such as a mouse, using human connective tissue connective tissue. Such connective tissue material from the human joint is obtained using methods known per se. The conjunctival connective tissue can be isolated by removing lymphocytes from the synovial tissue. The connective tissue is comminuted and the comminuted fabric is pressed through a stainless steel strainer. The connective tissue material is collected from the strainer.

The immunogen is administered in combination with an adjuvant, such as, for example, Freund's adjuvant deficient, although any suitable adjuvant may be used. The immunogen and adjuvant are injected using any known administration protocol or protocol that provides the induction of antibody formation. For example, the immunogen (a conjunctival connective tissue medium with approximately 1 pg VAP-1 antigen per injection) can be specifically injected three times per week into the patella of the patho-5 gene-free Baie / c mouse to induce an immune response.

The lymphocytes of the posterior lymph nodes are isolated using methods known per se, as described above, by squeezing the powdered lymph nodes through a stainless steel strainer and collecting the lymphocytes released from the strain through a strainer, then fused to non-10 18) using standard methods. The hybridomas can be screened using any suitable method, such as immunoperoxidase staining of glaciers. One hybridoma (1B2, subclass IgGi), which produced an antibody reactive with the vascular endothelium of the articular well, was cloned twice in limiting dilution and silenced for further studies. The antigen recognized by monoclonal antibody 1B2 was named VAP-1 (Muscular Adhesion Protein-1).

The antibodies used as VAP-1 binding proteins in the methods of the invention are monospecific antibodies, i. antibodies that specifically recognize only the VAP-1 protein or an antigenic portion thereof.

Monospecific antibodies can be both polyclonal and monoclonal.

Polyclonal antibodies can be prepared by inserting a substantially pure VAP-1 preparation into a suitable animal and then administering one or more supplemental injections at appropriate intervals.

However, in the methods of the invention, it is preferable to use monoclonal antibodies (or biologically active derivatives thereof, such as Fab ', F (ab') 2, or Fv fragments) recognizing VAP-1 antigen. It should be noted that monoclonal antibodies can be produced at any convenient site and thus may be, for example, murine or human monoclonal antibodies,

The antibody may also be produced by cloning the DNA sequence encoding the antibody or a biologically active derivative thereof into a suitable cell, e.g., a microbial, plant, animal or human cell, and culturing the cells under conditions favorable to the production of the antibody or its biologically active derivative. taking from culture the antibody of art or its biologically active derivative.

The antibodies of the reagent used in the present invention should preferably be in substantially pure form to improve the precision of the method.

7 Properties of VAP-1 5 Isolated VAP-1 has a mofecil weight of 90 kD under reduced conditions and 100 kD under non-reduced conditions.

The lymphocyte-free pharyngeal extract is a preferred source for the isolation of VAP-1 antigen. These extracts can be prepared by removing lymphocytes from the pharyngeal tonsil tissue by squeezing the comminuted tissue through a stainless ferric screen, the connective tissue material is recovered from the strainer. On the other hand, any cell type expressing VAP-1 can be used in the method described below, since in the exemplified methods, the final isolation is dependent on the affinity of VAP-1 and 1B2.

All steps should be performed in a cool (about 4 ° C) cell, gently disrupted (eg overnight at + 4 ° C) in a buffer containing proteolysis inhibitors, e.g., buffer containing 150 mM NaCl, 10 mM Tris-base, 0.15 mM MgCl 2 1% NP4Q, 1 mM PMSF and 1% aprotinin. These extracts can be pre-purified from debris caused by cell disruption using gentle centrifugation, e.g., 10,000 g for 30 min. The supernatant is then transferred to the affinity column system, in the following order, (1) normal mouse serum column, (2) non-specific fgGi antibody column (such as 1E12 or any commercially available non-specific IgG-1 antibody), and (3) monoclonal antibody 1B2 pole. The material bound to the 1B2 antibody column is eluted with 50 mM triethanolamine and matured. Using this approach, VAP-1 can be isolated from connective tissue of the tonsil. Other similar methods known per se may also be used.

Monoclonal antibody 1B2 shows that VAP-1 is abundant in HEV-like vessels in inflamed articular wells. VAP-1 is not present in infiltrating leukocytes or in any of the si-30 decidual components of connective tissue. In peripheral lymph nodes and tonsil, VAP-1 appears to be present in the majority of HEVs. VAP-1 is strongly localized to the iuminal side of the endothelial veins. Granular staining of VAP-1 is seen in the cytoplasm of endothelial cells as well as on the tissue-side surface.

Particularly in the tonsil, VAP-1 levels vary widely across 35 HEVs and a few individual HEVs (with typical rounded morphology) are completely VAP-1 negative. In appendix and in lamina intestinal proprias- 8 SA appears to be only a few faintly staining venules. Low expression VAP-1 also occurs in dendritic-like cells in germinal centers and in arterial, venous, and intestinal wall junctions in muscle cells.

In contrast, VAP-1 is almost completely absent from the 5 stacks of larger vessels. In addition to tissue necrosis lymphocytes, VAP-1 does not appear to be expressed in the following cells: peripheral blood lymphocytes, monocytes, NK cells, granulocytes and isolated laryngeal leukocytes, T-lymphoblastoid cell line CCRF-CEM (CCL-119), EBV transformed B cell line from E. Engieman, Stanford University), 10 monocytic cell line U937 (CRL 1593, ATCC); and leukemia cell lines KG-1 (CCL 246, ATCC); KG-1a (CCL 246.1, ATCC) and K562 (CCL 243, ATCC); endothelial cell line EaHy-926 (carcinoma and endothelial cell Hybridoma cells, Ed-Geli et al., Proc. Natl. Acad. Sci. USA 80: 3724 (1983)); as well as smooth muscle cells, fibroblasts and keratinocytes in prairie culture, and the epitheloid cell line He-15 La (CCL 2, ATCC). Human umbilical vein endothelial cells (HUVEC) isolated according to the Jaffe method (J. Clin. Invest 522745 (1973)) do not express VAP-1 as such, nor do they undergo 4 or 20 hours of IL-1 treatment (20-100 U / ml), TNF treatment. (200 U / ml) or LPS concepts (1.1, 1.0 pg / ml).

Comparison of VAP-1 with known endothelial cell molecules involved in leukocyte binding is known. Intracellular adhesion molecule I and 2 (10 AM-1 and ICAM-2), Vascular cell adhesion molecule 1 (VCAM-1), E-selectin (ELAIVi-1), and P-sextin (CD62, PADGEM, GMP 140) are all expressed in HUVEC- in cells either basally or after 25 inflammatory mediator induction (Springer, TA, Nature 346: 425 (1990); Stooiman, L.M., Cell 56.907 (1989); Osborn, Lt. Cell 62: 3 (1990); Pober and Cotran, Transplantation 50: 537 (1990); Butcher, E.C. Cell 67: 1033 (1991); de Fougerolles, A. R., et al., J. Exp. Med. 174, 253 (1991); Osborn L., et al. (1989) Cell 59: 1203; Bevilacqua MP, et al., Proc. Natl. Acad. Set. 84: 9238-30 (1987); McEver, R.P. et al., J. Clin. Invest 84: 92 (1989); Hattori, R., et al., J.

Biol. Chem. 264, 7768 (1989); Wellicome, S.M., et al., J. Immunol. 144: 2558 (1990); Pober, J.S., et al., J. Immunol. 137, 1893 (1986); Dustin, M.L., et al., J. Immunol 137, 245 ¢ 1986)). In contrast, VAP-1 is neither constitutively expressed nor induced by IL-1, TNF-alpha, or LPS treatment on HUVEC cells. The tissue distribution of these 35 molecules is clearly different, even when analyzing parotid sections of the tonsil (results not shown here), 1CAM stains 9 on most lumbar surface of large and smaller vessels and in some leukocytes (de Fougerolles, ARt et al., 17, Exp. 253 (1991); Dustin, M.L., et al., J. Immunol 137, 245 (1986)). On the other hand, VCAM-1 and ELAM-1 are stained only in a few veins of inflamed tissue (Rice, G.E., et al., J.

5 Exp. Med; 171, 1369 (1990); Rice, G.E. et al., Am. J. Pathol. 138: 385 (1991);

Cotran, R. S., et al., J. Exp, Med. 164, 661 (1986). In contrast, VAP-1 is strongly expressed in most HEVs at non-mucosal sites and is absent in all white blood cells and tested cell lines. Known adhesion molecules also differ markedly from VAP-1 in molecular weight, with the exception of ICAM-1 {IGAM-2 is 60 kD, VGAM-1 is 110 kD, E-selectin 115 kD and β-sextin 140 kD (Stooiman, LM. Cell, 56: 907 (1989); Osborn, L, Dell 62: 3 (1990); and de Fougerolles, ARt et al., J. Exp. Med. 174, 253 (1991)). In addition, VAP-1 is predominantly involved in lymphocyte binding, whereas CAMs, E- and β-sequin also are effective in the adhesion of polymorph-15 phonuclear leukocytes (Springer, TA, Nature 346, 425 (1990); Stooiman, L. iVL). , Cell 56, 907 (1989), Osborn, L, Cell 62, 3 (1990);

Pober and Cotran, Transplantation 50, 537 (1990); Butcher, EG, Cell 67, 1033 (1991)), The only human lymphocyte binding endothelial adhesion molecule described so far that is not expressed in HUVEC is the antigen recognized by the ME-20 CA-79 antibody (Berg, EL, et al. J. Cell Etoi 114, 343 (1991)). However, this is a tissue specific addressin of peripheral lymph nodes. In addition, VAP-1 is not present in all MECA-79 positive veins and purified B2 MECA-79 does not recognize purified MECA-79 antigen.

Thus, the expression pattern, function, and molecyl lineage of VAP-1 show-25 or that it is not identical to any of the lymphocyte binding lymphocyte binding pathways described above and that the degree of inflammation correlates with I

VAP-1 expression level in vivo. These results demonstrate that VAP-1 is relevant to understanding the physiological transport of lymphocytes in humans, and is a particularly valuable tool for studying the molecular mechanisms of tissue-selective lymphocyte deposition.

Use of VAP-1 and VAP-1 binding compounds

One embodiment of the VAP-1 binding compounds of the invention relates to reducing or treating inflammation in the human or animal body by in vivo administering to a human or animal subject in need thereof an effective amount of a VAP-1 binding compound (such as a VAP-1 reactive antibody). or a biologically active derivative of such antibody, or a soluble VAP-1 ligand).

The terms "treatment" or "treatment" are intended to encompass the administration of VAP-1 binding compounds to a subject for the purpose of prophylaxis, enhancement, prevention or treatment of conditions associated with VAP-1 protein adhesion. Specific VAP-1s prepared according to the present invention binding molecules include purified VAP-1 binding naïve and recombinant proteins such as antibodies or other molecules.

When administered to a patient, the reagents of the invention may be formulated in a manner suitable for parenteral, nasal, enteric or rectal administration. Thus, the reagents may be, for example, in injectable form, aerosol formulation, suspension, solution, bulk, etc. The reagent may be formulated using pharmaceutically acceptable additives or carriers such as isotonic saline in accordance with conventional pharmaceutical practice, and the dosage level of the reagent should be The anti-inflammatory effect of blocking -1 ;.

The reagent prepared according to the invention is suitable for the detection and treatment of any VAP-1 adhesion-associated elevated inflammatory response. Thus, the reagent is useful for identifying conditions such as arthritis, local infection, dermatosis, inflammatory bowel disease (IBP), and autoimmune disease.

In one embodiment, an effective amount of a VAP-1 binding compound is administered to provide therapeutic benefits over the deleterious secondary inflammation of the larynx. "An effective amount of" a VAP-1 binding compound means an amount by which the toxic effect of VÄP-1 mediated events is at least alleviated. An "elevated" host VAP-1 mediated event refers to a VAP-1 mediated adhesion patch in a subject that exceeds the medically healthy target. status standard. "Secondary-30" tissue destruction or toxic effect means the secondary tissue destruction or toxic effect that occurs in otherwise healthy tissue or organ and its cells due to increased VAP-1 mediated adhesion. This definition also includes events that occur due to "primary" excitement elsewhere in the body.

Infusion of a VAP-1 binding compound such as VAP-1 antibodies of the invention into a patient causes the binding of such antibodies to the surface of a patient's VAP-1-expressing cells, such as HEV, peripheral lymph node cells. HEV, and HEV of the pharynx, whereby the adhesion of these cells to other cells via VAP-1 is prevented and unwanted migration or migration of the lymphocytes into those tissues or cells is prevented. This prevents unwanted inflammatory responses due to VAP-1 mediated leukocyte transport and leukocyte migration from the bloodstream.

Thus, the pharmaceutical compositions prepared according to the invention are compositions containing VAP-1 binding compounds to such an extent that the binding of native VAP-1 to a biological VAP-1 target, and in particular to endothelial cells, is inhibited.

VAP-1 binding compounds can be conjugated, either chemically or by genetic engineering, to fragments of other agents that allow targeting of such a VAP-1 binding compound to a desired target.

Alternatively, other compounds may be conjugated to the VAP-1 binding compound either chemically or using genetic engineering to enhance this or add other properties to such a VAP-1 binding compound. In particular, it refers to properties that enhance the ability of a compound to promote VAP-1-mediated adhesion-mediated toxic activity.

The required doses and dosage forms of VAP-1 binding compounds can be readily determined by one of ordinary skill in the art having experience in the treatment of fungal diseases such as arthritis and tissue injury. In general, therapeutic doses of VAP-1 binding compounds will vary depending on the following considerations; the type of VAP-1 binding compound used; patient age; health-25 dentia; disease to be treated; other concomitant therapy, and possibly recurrence of treatment and desired type of response; frequency of tissue damage; the sex of the patient; duration of symptoms and possible contraindications tel other variables as defined by the attending physician. The desired dose may be administered in a single dose or in several doses according to the desired result. According to the invention, pharmaceutical compositions containing VAP-1 binding compounds which are subordinated to the invention may be in single-dose form.

Pharmaceutical compositions containing VAP-1 binding compounds prepared according to the invention may be administered in any suitable pharmacological carrier. They may be administered in any form that contributes to the prevention, amelioration, prevention or treatment of a VAP-1 mediated event in a human or animal. As used in the specification, the term "method of treatment" of the disease and the like are also meant to mean the prophylaxis of the disease in question.

The VAP-1 binding protein formulations of the invention for parenteral administration include sterile aqueous and non-aqueous solvents, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, fish oils and injectable organic esters. Aqueous carriers include e.g. water, aqueous alcoholic solutions, emulsions or suspensions comprising saline and buffered parenteral saline carriers such as sodium cionic acid solution, Ringer's dextrose solution, dextrose-sodium phosphorus solution, lactose-containing Ringer's solution or oils for intravenous use. liquid and nutrient liquids, electrolyte balancing agents, e.g. solutions based on Ringer dextrose and the like.

The VAP-1 binding compounds prepared according to the invention may also be administered in a pump-assisted or sustained-release form, particularly when the primary injury is delayed or prolonged rather than acute. An example of such a situation is an infection or fracture in which tissue or muscle damage is not noticed until several days after the event caused by the primary infection or injury, or if the infection or injury is permanent. VAP-1 binding molecules prepared according to the invention may also be administered to specific organs at high concentrations as part of a chimeric molecule (or complex) designed to be targeted to a particular organ.

Administration in sustained release form is particularly advantageous for the patient if repeated injections are required over a longer period. For example, it is preferred to administer the VAP-1 binding proteins of the invention in sustained release form when the methods of the invention are used to treat VAP-1 mediated hereditary or chronic inflammatory disease, thereby maximizing the therapeutic benefit for the patient.

The VAP-1 binding compound prepared according to the invention may also be used in dosage forms such as tablets, capsules, powders, or liquid solutions, provided that the biological activity of the VAP-1 binding compound is not impaired by gastrointestinal absorption and .

The process for preparing the pharmaceutical compositions of the invention may include methods known per se, e.g., conventional mixing, granulating, granulating, dissolving, freeze-drying or the like. The compositions of the invention are useful as such in the control of chronic or acute VAP-1 induced physiological damage. The compositions prepared according to the invention resist the maximal efficacy of the mechanism that recognizes the body's own VAP-1 activation.

In an intravenous dosage form, the compositions of the invention exhibit a sufficiently rapid action to be useful in the acute treatment of potential tissue damage.

In addition, the low-acting form is useful for the treatment of mild or chronic VAP-1 mediated conditions.

VAP-1 binding proteins which are substantially free of natural contaminants can be isolated and purified from natural sources or recombinant sources using standard known conditions and methods used to isolate such proteins, such as extraction, precipitation, chromatography, affinity chromatography, .

The following examples are intended to illustrate the present invention and in no way limit the scope of the invention.

EXAMPLE 1 VAP-1 Tissue Distrophy Histology VAP-1 tissue distribution was determined using immuno-20 superoxidase staining of ice cubes. The sections were incubated with primary antibodies (1B2 and 3G6 as control, mouse IgGI-Vasia against chicken T cells) for 30 min. The phosphate buffer was washed twice with physiological saline (PBS, 8 g NaCl, 1.21 g K2HPO4 and 0.34 g KH2PO4 per liter, pH 7.2), followed by addition of peroxidase-conjugated sheep anti-quin IgG (Dakopatt, Denmark) 5% AB-. In PBS containing 25 sera for 30 min. Next, 33 L-diaminobenzidine hydrochloride was used as chromogen in RBS containing 0.03% hydrogen peroxide. After staining, sections were counterstained with hematoxylin. For immunofluorescence staining, 3 µm ice sections were covered with primary antibody and FITG-conjugated fampa antf mouse IgG (Sigma, 30 St. Louis) was used as a second-stage reagent.

Immunohistological staining showed that monocionic antibody 1B2 stained the HEV-ectopic veins in heavily inflamed synovial wells (Figure TA), and no staining was observed in the infiltrating leukocytes, as did in the inflamed nivefestroma. The antigen recognized by monoclonal antibody 35B was designated VAP-1 (Eng. Vascular Adhesion 14

Protein-1). In peripheral lymph nodes and tonsil, mAb 182 reacted with almost all HEVs (Figure IB). VAP-1 was expressed strongly on the luminal side of endothelial cells (Figure 1C). The cytoplasm of endothelial cells showed granular staining and also the tissue-side surface was mAh 1B2 po-5 positive, There was a marked variation in staining intensity between the different HEVs, and a few bulging HEVs were 1 B2 negative (Figure 1B). In appendix and in lamina propria of the gut, only few faintly staining venules were detected (Figure 10). Poor expression of VAP-1 was also seen in germ-like cells like dendritic cells and on the surface of arteries, veins and smooth muscle mass of the intestinal wall. On the other hand, VAP-1 was almost completely absent from the luminal sides of large vessels. As well as tissue section lymphocytes, peripheral blood lymphocytes, monocytes, NK cells, granulocytes, and laryngeal lymphocytes were all completely 1 B2 negative in FACS analyzes. VAP-1 was absent from T lymphoblastoid (CCRF-CEM), B-15 lymphoblastoid (KCA, IBW-4), monocyte (U937) and leukemia (KG-1, KG-1a, K 562) cellular wells. In addition, VAP-1 was absent from human umbilical cord endothelial cells (HUVEC) in the basal cell; and 4 h or 20 h treatment with IL-1 (20,100 U / ml), TNF-β (200 U / ml) or LPS (0.1, 1.0 pg / ml) did not induce its synthesis. One endothelial cell line (EaHy-926) was also 1B2 negative. Smooth pleura, fibroblasts and keratinocytes that were overexpressed in primary culture and one epithelial (He-La) cell line did not express VAP-1.

Example 2 Intracellular localization of VAP-1

In addition to the tissue localization assay described in Example 1, intracellular localization of VAP-1 was also examined by confocal microscopy of the tonsil-thick sections (15 µm). The sections were incubated for 15 min with mAb 1B2 or 3G6, washed twice with PBS and covered with FiTC-conjugated sheep anti-mouse Ig for 15 min. The samples were then covered with glycerol-PBS solution using phenylene diamines as an anti-fadeing factor before confocal microscope-30. Microscopic examination clearly showed that VAP-1 is present on the luminal surface of HEV and in separate cytoplasmic granules. The identity of these granules is so far unclear, but in two-color immunofluorescence fluorescence with 1 B2, the granules did not co-localize. Thus, VAP-1 positive granules do not take up endothelializations

Weibel-Palade bodes known to contain the endothelial cell P-setectin molecule.

15

Example 3 Determination of the molecular weight of VAP-1

Lymphocyte-free throat extracts were lysed in buffer (150 mM

NaCl, 10 mM Tris-base, 0.15 mM MgCl 2, 1% NP 4 O, 1 mM PMSF and 1% aprotinin) overnight at 4 ° C. The lysate was centrifuged at 10,000 g at 4 ° C. The supernatant was pre-purified on a Sepharose CL-4B column (Pharmada, Sweden). The lysate was then passed through three CNBr-activated sepharose columns (Sepharo-10 se ~ 4B, Pharmacia) derivatized with either normal mouse serum, irrelevant IgGi antibody or 182 antibody (5 mg / ml, 5 mL column). The columns were washed thoroughly with lysis buffer. The material bound to the 182 column was then eluted using 50 mM triethanolamine, cold-flowed, dissolved and subjected to SDS-PAGE (7.5%, reduced) and visualized by silver staining.

For iodine staining, lymphocyte-free throat extracts were digested in RPMI 1640 containing 100 U / ml collagenase (Clostridium histolytjcum, type li, Sigma), 10% FCS, antibiotics and 10 mM Hepes for 1 hour at 37 ° C with gentle agitation. Following collagenase treatment, cells were washed with HBSSr and surface-labeled using the 12SI and lactoperoxidase method. The iodine-labeled cells were lysed in buffer and the lysate was pre-purified by centrifugation at 10000g for 15 min. The lysate was further purified for 16 hours at 4 ° C with CnBR-activated Sepharose coupled to normal mouse serum. Immunoprecipitation was performed using CnBR-activated 1B2 or 306 coated Sepharose 4B beads. Samples were analyzed using 7.5% SDS-PAGE under reducing conditions (2-mercaptoethanol).

The molecular weight of VAP-1 was determined from the tonsil using an affinity-tagged molecule by SDS-PAGE. Silver staining of the gel indicated the predominant band of 90 kD molecular weight under reduced conditions (Figure 30 2). VAP-1 moved slightly slower under non-reduced conditions (Mr 100 kD).

Analysis of immunoprecipitates of iodine-stained laryngeal tissue confirmed the reactivity of 1B2 antibody with the 90 kD molecule (and with a slightly smaller degradation product (Figure 2). In some cases, a 180-200 kD band was also visible.

16

Example 4

Binding of lymphocytes to the pharynx, peripheral lymph node (PLM), articular cavity, and HEVr of the appendix, and the granulocyte binding to HEV 5 The details of this method were previously published in Jalkanen and Butcher, Blood 66, 577 (1985). Briefly, freshly cut sections of human tonsil, articular cavity, appendix and peripheral lymph nodes were incubated with 1B2 or 3G6 supernatant for 80 min at 7 ° C with gentle rotation. Ficoil-isolated lymphocytes (3x106 / well) in 5% FCS and 10 mM Hepes were added and incubated for 30 min. after incubation, non-adherent cells were carefully discarded and adherent cells were fixed overnight in cold PBS containing 1% glutaraldehyde.

HEV-bound cells were counted from 4 to 6 slices per tissue and sample (at least 100 HEV) on a single-blind basis. A similar procedure was used to determine granulocytes, except that the granulocytes (isolated using Histopaque 1119, Sigma) were kept in Ca 2 * and Mg 2+ free HBSS until transferred to sections.

Tissue distribution of VAP-1 in endothelial cells in vivo indicated that the protein may act as a specific recognition element for leukocytes. Therefore, the functional role of VAP-1 in HEV binding was studied using a modified Stamper-Woodruff in vitro method (Jalkanen and Butcher, Blood 66, 577 (1985)). Pretreatment of ice sections with 1B2 inhibited lymphocyte binding to HEV (Fig. 3), the inhibitory effect was most pronounced in the tonsil and peripheral lymph nodes, but also the binding to the HEV of the cerebellum was significantly reduced. The effect of lymphocyte binding on endocrine HEV and of granulocytes on laryngeal HEV was less (Figure 3). These results indicate that VAP-1 either contributes to or is closely related to endothelial cell factors involved in the recognition of lymphocytes from peripheral lymph nodes, the tonsil and the articular cavity. For the direct evaluation of the association of VAP-1 with Jymfo-30 in the cytoplasmic endothelial interaction, the binding of lymphocytes to the affinity-purified VAP-1 protein was studied (Figure 4).

Lymphocytes efficiently adhered to well-bound VAP-1. Lymphocyte binding to VAP-1 was specifically inhibited by the 1B2 antibody but not the 3G6 control antibody. Monoclonal antibody 182 did not inhibit the binding of fym-35 phosphites to another non-relevant endothelialium molecule (Figure 4).

i 17th

Tissue distribution and HEV binding indicate that VAP-1 is primarily involved in the transport of lymphocytes to peripheral lymph nodes, the tonsil and the synovium. Interestingly, lack of VAP-1 expression in the tonsil defines a small postcapillary venous population that is not morphologically different from 1 B2-positive. Because of its close connection with the intestine, the pharynx may have both HEVs of the lymphoid phage (VAP-1 negative) and peripheral lymph nodes (VAP-1 positive). It remains to be determined how phenotypic differences in VAP-1 expression correlate with the binding capacity of individual HEV lymphocytes. Small amounts of VAP-1 in mucosal lymphoid organs suggest that this endothelial antigen may be subject to differentiated reguftion in various lymphocyte recognition systems. In addition, the level of inflammation correlates with VAP-1 expression in vivo.

Example 5 Effect of 15 1 B2 * on Cell Binding to VAP-1 The affinity of VAP-1 and 1E12 was purified from the pharyngeal extracts as described in Example 2. Purified VAP-1, 1E12 and heat-inactivated BSA were diluted in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM MgCl 2) 2 mM CaCl 2 containing 0.01% β-octylglycopryanoside as a detergent. The protein was absorbed into 20 glass wells (Lab-Tek well plates, Nunc) for 16 h at + 4 ° C. PBS containing 1 mg / ml BSA was blocked for 30 min at room temperature, then 1B2 or 3G6 supernatants were added to the wells and incubated for another 30 min at room temperature. In addition, freshly isolated peripheral blood mononuclear cells were incubated in RPM11640 with 10% FCS and 10 mM Hepes for 1 hour at 37 ° C in tissue culture flasks to remove monocytes. Non-adherent lymphocytes (1.8x106 wells / well) 100 pf RPM1 1640: 858 were added to each well. After incubation for 30 min at 37 ° C, the non-adherent cells were discarded. The wells of the wells were removed and the plates were washed in a fine stream of RBS, fixed in cold PBS containing 1% glutaric dehyde. Cells were then stained using 30 Diff-Quick dyes. The adherent cells were quantitated visually by counting the number of cells in each well (total area 50 mm 2 / sample).

18th

Example 6 Partial amino acid sequence of VAP-1 protein The partial amino acid sequence of the 90 kD VAP-1 antigen was determined. This partial amino acid sequence is 20 amino acids in length: 5 TEDGDMXLVNGASANEGXVE [SEG iD NO. : 1:]. A search of databases of protein and DNA * sequences showed that this sequence does not belong to any of the previously known sequences.

Example 7 VAP-1 Assay in Clinical Specimens Demonstrating Inflammatory Response Fresh samples of normal larynx, peripheral lymph nodes, intestine and heart were obtained from surgery and kidney specimens were obtained from non-donors. Other tissues are derived from biopsy specimens. All samples were histopathologically determined as non-inflammatory. Inflammatory specimens were obtained from skin biopsies (psoriasis, atopic dermatitis, lichen ruber pianus) of patients with chronic dermatosis. Control biopsies were taken from the same patients in macroscopically defined areas. Intestinal inflammatory specimens were obtained from patients with inflammatory bowel disease (Crohn's disease, ulcerative colitis) who underwent therapeutic surgery. Samples of articular wells were derived from synovectomies.

Samples were stained with immunoperoxidase as described in Example 1. Briefly, acetonized sections of ice were incubated with a primary antibody (culture supematantil or 50 pg / ml purified immunoglobulin) and a suitable peroxidase conjugate with a second-stage reagent and a color reaction was performed using H2O2 and diaminobenzidine as substrate. Expression of VAP-1-25 in intestinal and skin samples (normal and inflamed) was analyzed from coded samples by two independent investigators without knowing the diagnosis.

As noted in Example 1, VAP-1 is expressed only at low levels in some normal non-inflamed gut veins (Figure 5A). On the other hand, a significant increase in VAP-1 expression was observed in the intestinal sample of a patient with inflammatory bowel disease (Figures 5B and 5C and Table 1). VAP-1 was induced both in thin-walled veins of Lamina propria and in HEV-like veins of the organized lymphatic follicle (Peyer's patches). Increased synthesis of VAP-1 was also correlated with chronic inflammation of the skin (Figures 6A and B). To exclude individual differences affecting the results, 35 simultaneous samples of dermatosis lesion from the same patient and 19 control samples from healthy skin were taken. Also in these samples, the number of VAP-1 positive veins in the upper dermis was higher in the inflammatory sample than in the corresponding control area sample. In addition, VAP-1 positive veins were always associated with significant penovascular leukocyte-5 infection.

2d

Table 1 Induction of VAP-1 in Gingivitis Disease Sample n VAP-1 - + 4- + +++ ++++

Normal 9 0 5 3 1 0 C röhit 7 0 0 2 3 2

Ulserat, moth. 7 - 0 0 0 5 2

Intestinal samples were taken from surgical patients with orohn disease, ulcerative colitis, and tumors (Normal areas of tumor samples represent "normal" samples).

The number of VAP-1 positive veins per sample was determined, ++++ as described above.

Example 8 VAP-1 contributes to an inflammatory mucosal clearance reaction

An in vitro ice-up binding assay was performed as described in Example 3, Monoclonal antibody 1B2 inhibited lymphocyte binding to small vessels of Lamina propria by about 60% (Figure 7) in two intestinal samples where VAP-1 expression was significantly suppressed.

As the invention has been described herein with reference to specific use examples, it should be understood that it may be further modified and this application is intended to cover any variation, use or adaptation to the invention which follows the principle of the invention, 20 to the known or conventional use of the subject matter of the invention and applicable to the essential characteristics described above to the extent required by the appended claims.

21

Sequence Identification (1) GENERAL INFORMATION: (i) SEARCH: (A): NAME: Jalkanen, Sirpa (8): STREET: Rauvolantie 79 (C): CITY: Bishop's Cross

(E): COUNTRY: FINLAND

(F): POSTAL NUMBER: FI-2Ö76Q

(i) SEARCH: (A): NAME: Salmi, Marko (8): STREET: Keilonsoittajankatu 8a A13 (C): CITY: Turku

(E): COUNTRY: FINLAND

(F): POST NUMBER: FI-2050G

(ii) NAME OF THE INVENTION: Human Endothelial Cell Molecule Affecting Lymphocyte Binding (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) TO: Biotie Therapies Oy] (B) Street: Tyk:

(E) COUNTRY: FINLAND

(F) POSTAL NUMBER: FI-20520 (v) PUNCH CODE FORMAT: (A) MEDIA TYPE: Floppy (B) COMPUTER: Fujitsu Siemens (C) OPERATING SYSTEM: M / S Office 22 (D) SOFTWARE: Microsoft Word (vi) CURRENT SEARCH (A) APPLICATION NO: 945784 (B) APPLICATION DATE: June 9, 1993 (2) SEQ ID NO: 1 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 20 amino acids (B) TYPE (C): amino acid TOPOLOGY: linear (ii) MOLECYL TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQUENCE

NO.1:

Thr Glu Asp Gly Asp Met Xaa Leu Vai Asn Gly Ala Ser 1 5 10

Area Asn Ghu Gly Xaa Vai Glu 15 20:

Claims (11)

23 1. Substantially purified and isolated human endothelial vascular adhesion protein, VAP-1, feeling 11 u that the VAP-1 is recognizable by the help of a monoclonal antibody produced by the hybridoma cell line of DSM AGO 2041. through. VÄP-1 according to claim 1, characterized in that said VAP-1 has a molecular weight of 90 kDa. VAP-1 according to claims 1-2, characterized in that said VAP-1 binds to lymphocytes. VAP-1 according to claims 1-3, characterized in that said VAP-1 is produced by binding said VAP-1 to an anti-VAP-1 antibody. 5. Hybridoma cell line, characterized in that it produces anti-VAP-1 antibody and has been assigned accession number DSM ACC2041. A method for producing an anti-VAP-1 antibody or anti-VAP-1 binding fragment thereof, which specifically recognizes a human endothelial VAP-1 protein according to any one of claims 1 to 4 and which the antibody or binding fragment does not bind to human neoplastic human chimpanzee (HUVEG) surface proteins, it being understood that the method comprises producing the antibody or binding fragment either in a hybridoma cell line or in a cell cloned with the antibody or binding fragment. 7. The method of claim 6, wherein the method produces a monoclonal antibody 1B2 that specifically recognizes the vessel adhesion protein VAP-1. A process for the preparation of a cell-free composition comprising an anti-VAP-1 antibody or an anti-VAP-1 binding fragment thereof, characterized in that it comprises a method according to claim 6 or 7 as well as cell removal. A process for the preparation of a pharmaceutical composition comprising an anti-VAP-1 antibody or an anti-VAP-1 binding fragment thereof, characterized in that it comprises the process of claim 6 or 7 and the formulation of the composition for injection. A method according to claim 9, characterized in that the composition is formulated in a unit dosage form. 24 11. A method of inhibiting VAP-1-mediated endothelial bone binding to lymphocytes, wherein 11 µl of VAP-1-mediated lymphocyte endothelial cell adhesion reaction is inhibited by administering a sufficient amount of the VAP-1 binding compound prepared according to claim 6, 8 or 9. endo-5 to bioclave the VAP-1 binding sites involved in this reaction. 25