JPH07506244A - Antibody that recognizes the fourth immunoglobulin-like domain of VCAM1 - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] VCAMI fourth immunoglobulin-like Domain Tongue Blade and y to λ technique J ro Abuno The present invention relates to monoclonal antibodies (MAbs), specifically vascular cell adhesion molecule-1 (VC The present invention relates to a monoclonal antibody that binds to domain 4 of AMI.

1 free prisoner VC-AMI (also known as INCAM-110) is an inflammatory cytokine. Adhesions induced in endothelial cells by In (TNF and IL-1) and LPS. was first identified as a child (Rice et al., 1989 [1], Ospone et al., 1 989 [2 pieces). VCAMI includes T and B lymphocytes, monocytes and eosinophils binds to cells expressing integrin VLA-4 (α4β1) but not neutrophils. and is involved in recruiting these cells from the bloodstream to areas of infection and inflammation. (Elysis et al., 199o [3]; 7j Spawn, 1990 [4] ), the VCAM1/VLA-4 attachment pathway is involved in many physiological and pathological processes. Related. VLA-4 is generally restricted to hematopoietic lineages, but melanoma cell lines Therefore, VCAMI is involved in the metastasis of this type of tumor. has been suggested (Rice et al., 1989 [1]).

In vivo, VCAMl has been shown to induce early atherosclerosis in a rabbit model system. found in areas of arterial endothelium that exhibit inflammatory plaques (Cybulski and Gimbron) , 1991 [5]), and VCAMl is also found in vesicular cells in human lymph nodes. It is also found in process cells (Friedman et al., 1g90 [6]). Additionally, this Also present in bone marrow stromal cells in mice (Myake et al., 1991 [7]), VCAMI therefore appears to play a role in B-cell development.

VCAMI belongs to the immunoglobulin (Ig) superfamily. herein The main form of endothelial cells, called VCAM-7D, consists of 7 Ig homologs. It has a logical unit or domain. Domains 4.5 and 6 are amino acid sequences similar to domains 1.2 and 3, respectively, in the history of gene development. suggesting an intergenic duplication event (Osporn et al., 1989 [2], Porte et al. 1990 [8]; Hetsion et al., 1991 [9]). Additionally, other splices A slight 6-domain type (herein referred to as VCAM-6D) generated by ), in which the fourth domain is deleted (Ospone et al., 1989). [2], Hetsion et al., 1991 [9]; Cybalski et al., 1991 [ 10]). The biological significance of this alternative splicing is unknown, but VCAM-6D can bind to VLA-4-expressing cells as shown in It therefore clearly has potential functionality in vivo.

Infection, inflammation, and risk of VCAMI/VLA-4 in atherosclerosis. The clear involvement of the adhesion pathway indicates the mechanism of cell-cell adhesion at the molecular level. Researchers have continued extensive research to understand this mechanism, and researchers are considering interventions in this attachment pathway. It has been proposed as a treatment for qi (especially inflammation) (Ospone et al., 1989 [2 pieces]).

Monoclonal antibodies that block VCAMI binding to VLA-4 are known.

For example, anti-VLA-4 MAbs HP2/1 and HPI/3 are VLA-4 expression on cells and VCAMI-transfected CO8 cells has been shown to prevent Ramos cell adhesion (Elysis et al., 1990). [3]).

Furthermore, for example, the monoclonal antibody 4B9 (Carlos et al., 1990 [11 Co.) Anti-VCAMI antibodies such as Ramos (B-cell analogue) against RUYO-transfected CO8 cells , Jurkat (T-cell-like) and HL60 (granulocyte-like) cell attachment (Hession et al., 1991 [9]).

Although blocking antibodies that may be potential therapeutic agents are also known, other non-binding antibodies of VCAM1 are known. A novel monoclonal antibody that recognizes an epitope in the VLA An important research tool to map the parts of VCAMI that are critical for binding to -4. becomes. For example, without competing with the epitope of a blocking antibody such as 4B9 (V Antibodies that bind CAMI also have important diagnostic uses. For example, VLA-4 binding using a radiolabeled anti-VCAMI antibody that recognizes an epitope not essential for The VLA-4-binding domain of VCAM1 can be used to monitor the process and location of acute inflammation. It will not inhibit the action of subsequently administered therapeutic agents that interact with the main.

λ匪台IA This time, the present inventors bind to VCAM-7D but not to VCAM-6D, Therefore, it is likely that a group of Monoclonal antibodies were isolated. Surprisingly, the regulations for these domain 4 antibodies are found to block Ramos binding to VCAM-7D-expressing cells. Therefore, by using these antibodies, domain 4 of VCAM-7D was isolated from MAb4B9 (domain In addition to the regions recognized and blocked by main 1), there are also regions involved in VLA-4 binding. It was also shown that the Therefore, VCAMI is a single receiver (VLA -4) was identified as the first known adhesion molecule with two binding sites for , domain 4 monoclonal antibodies according to the invention map the molecular functionality of VCAMI. (and at least partially block VCAMI recognition by VLA-4) It is also useful for

Therefore, the object of the present invention is to recognize VCAM-7D but not VCAM-6D. The objective is to provide monoclonal antibodies that do not.

Furthermore, it is an object of the present invention to obtain the fourth immunoglobulin-like domain (“domain”) of VCAM-7D. Antibodies that recognize epitopes (“domain 4 antibodies”) that are dependent on provides a domain 4 antibody that does not cross-block anti-VCAMI MAb 4B9. It's about doing.

A further object of the present invention is to block the adhesion of VLA-4-expressing cells to VCAMI. The object of the present invention is to provide a domain 4 antibody that can be locked.

These and other objectives are achieved by the monoclonal antibodies disclosed herein. be done.

A day of Figure 1 shows the linear arrangement of functional domains from the amino terminus to the carboxyl terminus. 1 is a schematic diagram of VCAM-7D shown in FIG. These are signal peptide (rsigJ), immune Globulin-like domains 1-7, transmembrane sequences (rtmJ) and cytoplasmic domains, all That is, [tail J (rcytj). In addition, the phases of several restriction enzyme recognition sites are Counterpositions are also shown and these sites can be used to modify one or more modified domains ( Other VCAMI constructs were made with (described below). In the examples described later C0 using cDNA encoding VCAM-7D with the configuration shown. By transfecting 87 cells, VCAM-7D was displayed on their surface. COS7 cells were produced.

Figure 2 shows a linear arrangement of functional domains from the amino terminus to the carboxyl terminus; i.e. signal peptide (rsi gJ), immunoglobulin-like domain 1.2 ．． 3.5.6 and 7, transmembrane sequence (rtmJ) and cytoplasmic domain, i.e. ``This is a schematic diagram of VCAM-6D showing Tail J (rcytJ). In the example, a cDNA encoding VCAM-6D with the configuration shown is used. By transfecting CO37 cells with VCAM- C087 cells exhibiting 6D were generated.

Figure 3 shows the chimera ■CA-M1 (VCA This is a schematic diagram of the M/I C-AM-1. This polypeptide is VCAM -7D, but the cDNA encoding VCAM-7D is The segment encoding most of domain 1 and part of domain 2 was excised. It was created using a cDNA encoding a similar portion of intercellular adhesion molecule-1 (ICAMI). Modified so that it can be replaced. Modified portions of the VCAM-7D molecule are indicated by shading.

Figure 4 shows another texture-7 VCAM1 (VCA FIG. 1 is a schematic diagram of the M/ICAM-2. This polypeptide is VCAM-7D has the general structure of , except that most of domain 1 is and the cDNA encoding a large portion of domain 2 was excised to create ICAMI Replaced by cDNA.

FIG. 5 shows another chimeric VCAMI (VCAM /ICAM-3). This polypeptide is VCAM-7D It has a general structure, but with a portion of domain 1 as shown by the shaded area. c encoding all of domains 2 and 3 and a small N-terminal portion of domain 4 The DNA was excised and replaced with ICAMI cDNA.

Figure 6 shows cDNA encoding VCAM-6D or VCAM-7D. Expression in CO37 cells transfected with any of the following cDNAs: It is a graph showing the relative level of. VCAM-6D or VCAM-7D For the present, flow cytometry after indirect immunofluorescence staining with MAb 4B9 Analyzed by fluorometry. Control transfectant (pCDM8 vector The number of cells with a fluorescence intensity greater than that indicated by 99% of the Decided. Data shown are the combined results of two experiments, standard deviations are indicated by error bars. be done.

Figure 7 shows VCAM-7D, VCAM-6DSVCAM/ICAM-1, VCA M/I by cDNA encoding CAM-2 and VCAM/ICAM-3 The extent of Ramos cell binding to separately transfected CO8 cells This is a graph showing. Furthermore, 20 μg/ml MAb 4B9 (anti-VCAM I) and 10 μg/ml MAb HPI/2 (anti-VLA-4). The effect of RamO8 binding on pre-incubating transfected CO8 cells is shown. moreover , cells transfected with pCDM8 (expression vector only; negative control) and and a negligible effect on cells transfected to exhibit surface I CAMI. The Ramos binding is also shown. The connection is with Ramos cells connected per 1 mm2. (in the figure, "bound Ramos cells/mmJ").

FIG. 8 shows another structure of VcAM1 (VCA M-6D/ICAM-1). This polypeptide is VCAM -6D general structure (no domain 4), but as indicated by the shaded area The cDNA encoding most of domain 1 and a small portion of domain 2 is excised. and replaced by ICAMI cDNA.

Figure 9 shows chimeric CAMI (VCAM-6 D/ICAM-2). This polypeptide is VCAM-6D (without domain 4), but with domain 4 as indicated by the shaded area. The cDNA encoding most of domain 1 and a large portion of domain 2 has been excised. Replaced by rcAMlcDNA.

Figure 10 shows VCAM/ICAM-1, VCAM'-6D/ICAM-1, VC cDN encoding AM/I CAM-2 and VCAM 6D/ICAM 2 Ramos cell binding to CO8 cells separately transfected with A. It is a graph showing the degree of Furthermore, cells transfected with pCDM8 (expression vector only; negative control) and transfected to show surface ICAMI. It also shows negligible Ramos binding to transfected cells.

FIG. 11 shows another chimeric VcAM1 (VCA It is a schematic diagram of M-6D/VCAM4-1 (!). This polypeptide is V Has the general structure of CAM-6D (without domain 4), but indicated by the shaded area The cDNA encoding domain 1 and a small portion of domain 2 is excised as follows. domain 4 (and one amino acid of domain 5) of VCAM-7D. was replaced by a cDNA encoding

Figure 12 shows VCAM/I CAM-1, VCAM-6D/ICAM-1 and Transfected separately with cDNA encoding VCAM-6D/VCAM4-1. FIG. 2 is a graph showing the degree of Ramos cell binding to infected CO8 cells. . I Negligible Ramos for CAMI and negative (pCDM8) controls Also indicates a bond.

Figure 13 shows the presence of inhibitory antibodies or MAb 4B9 or MAb GH. 12 (invention) expressing various chimeric VCAMI constructs. 1 is a graph showing the degree of RamoS cell binding to transfectants.

Figure 14 shows the presence of inhibitory antibodies or MAb 4B9 or MAb ED. Tigers expressing various chimeric VCAMI constructs in the presence of II (invention) 1 is a graph showing the extent of Ramos cell binding to transfectants.

FIG. 15 shows the presence of inhibitory antibodies or MAb 4B9 or MAb GE Transcripts expressing various chimeric VCAMI constructs in the presence of 4 (invention) FIG. 2 is a graph showing the degree of Ramos cell binding to the sfected body.

Figures 16.17.18 and 19 show that 20 ng/ml recombinant human TNF 4 hours (Figure 16), 24 hours (Figure 17), 48 hours (Figure 18) and 7 hours Ramos cell binding to 1tJVEC (culture treated for 2 hours) This is a graph showing. Uninduced HUVEC (control), induced (TNF-treated) HUV EC only, HPI/2 (anti-VLA-4α4 MAb), 4B9 (anti- VCAMI MAb), EDII (anti-VCAMI MAb, the present invention) and and Ramos cells in the presence of GH12 (anti-VCAMIMAb, invention) Compare the bonds of.

5B of a Techniques for producing monoclonal antibodies are well known. In short, the immortal cell line ( (typically murine myeloma cells) expressing a given antigen (e.g. VCAMI). Lymphocytes from mammals immunized with whole cells and/or purified antigens ( The culture supernatant of the resulting hybridoma cells is incubated with anti-inflammatory cells (typically tile cells). screen for antibodies to the antigen (generally Cora and Milstein). , 1975 [12]).

Immunization can be performed using standard methods. Unit dose and immunization mode depends on the type of mammal being immunized, its immune status, the weight of the mammal, etc. Ru.

Typically, the immunized mammal is bled and the serum from each blood sample is appropriated. Analyze for specific antibodies using a screening assay. For example, - For vCAMI antibodies, recombinant soluble VCAM-7D (rsVCAM- Ability of immune serum to block Ramos binding to plates coated with 7D) It was identified by testing. Typically used to produce hybridoma cells Lymphocytes are detected by this type of screening assay for the presence of anti-VCAMI antibodies. isolated from an immunized mammal whose serum has already been tested positive. .

Typically, immortal cell lines (e.g. myeloma cell lines) are from the same species as lymphocytes. Obtained from mammals of the same species.

Preferred immortal cell lines contain hypoxanthine, aminopterin and thymidine This is a mouse myeloma cell line that is sensitive to the medium (rHAT medium). .

Typically, HAT-sensitive mouse myeloma cells are incubated with polyethylene glycol ( For example, PEG 3350) is used to fuse to mouse tile cells. Then gain from fusion The resulting hybridoma cells are selected using HAT medium, which contains unfused and kills nonproductively fused myeloma cells (unfused tile cells are transformed) (It will die after a few days). Hybridomas that produce the desired antibodies are Hybridoma culture supernatants were screened by analysis to detect MAbs with the specificity of Lean and detect. For example, the hybridoma according to the invention may be VCAM- Capable of binding to 7D-expressing cells but not VCAM-6D-expressing cells. (Example 1, see above).

This type of screening assay results in the production of anti-VCAMI antibodies. to cause tested hybridoma cells to secrete monoclonal antibodies into the culture medium. Cultured in nutrient medium under sufficient conditions and time. Suitable for hybridoma cells Tissue culture techniques and media are well known. On hybridoma culture without cells The clear liquid can be collected, and the anti-VCAMI antibody can be further purified by well-known methods if necessary. can.

Alternatively, hybridoma cells can be treated with 2.6,10.14-tetramethylpentadecane. (Bristin; Sigma Chemical Company, St. Louis, MO) It can also be injected intraperitoneally into trained mice to produce the desired antibodies. C Ibridoma cells grow in the peritoneal cavity and secrete antibodies, which accumulate as vaginal fluid. do. This antibody can be recovered by withdrawing the fluid from the abdominal cavity with a syringe. .

The monoclonal antibody of the present invention is a 7-Ig-domain type (7) VCAMI (t). ゎchiVcAM-7D) (7) Recognizes epitope dependent on the 4th 1g domain Ru. That is, the MAb of the present invention binds to the fourth 1g domain of VCAM-7D (7). binds to an epitope contained or partially contained in the fourth 1g domain, Removal of the fourth Ig domain eliminates the ability to recognize VCAMI structures. these The MAb is referred to herein as [domain 4 MAbJ].

Domain 4 MAb binds to VCAM-7D- but not to VCAM-6D do not. All antibodies of the invention are known VCAMI/VL antibodies such as 4B9. Different from A-4 block antibody. In this application, 4B9 is the domain of VCAMI. VCAM-7D and VCAM-6. D: It is shown that both sides are recognized. The monoclonal antibody of the present invention is VCA Recognizes M-7D but does not cross-block MAb 4B9. described here The monoclonal antibody regulation is based on Ramos cells against VCAM1-expressing cells. and other VLA-4-expressing cells. Recorded here The regulations for the domain 4 monoclonal antibody listed are for mononuclear leukocytes and VCAMI-producing VCAMI that does not participate in the VCAMI-mediated adhesion pathway between existing cells binds to the epitope of antibodies of the latter type), for example VCAMl (particularly VC AM-7D) to inhibit VCAMI binding in vitro or in vivo. Useful for detection without harm.

Monoclonal antibodies of the invention and other anti-VCAMI antibodies obtained from other researchers Using MAb, binding of VLA-4 to VCAMl was determined by binding of domain 1 to VCAMl. scales occurring at two different sites, the other requiring domain 4. I figured it out. The definition of anti-VcAMI domain 4 MAb according to the present invention is -VCA in cultured endothelial cells with the ability to block 4-dependent cell binding The fact that binding through domain 4 may also block cellular binding to MI is an important functional part of the VCAMI/VLA-4 interaction in vivo. and

The monoclonal antibody of the present invention can be produced naturally as described above, or Alternatively, it can also be synthesized using recombinant DNA technology. Suitable recombinant antibodies may be For example, host cells containing DNA encoding the immunoglobulin light and heavy antibodies of the desired antibody. Antibodies and recombinant chimeric antibodies produced by transformation with suitable expression vectors Here, the heavy chain and/or light chain of the anti-VCAMI antibody Some or all of the constant, constant and/or variable regions may be used in different types of immunoglobulins. Substituted with the corresponding region of Robulin light chain or heavy chain. This is the so-called "human body-compatible” antibodies (e.g., Jones et al., 1986 [13 Co.; Do et al., 1989 [14] and US Pat. No. 4,816,397 (Boss et al.) [ 15], all of which are hereby incorporated by reference).

Furthermore, for example Fab, Fab', F(ab)2 and F(v) fragments domain 4-binding fragments of anti-VCAMI antibodies such as; heavy chain monomers or dimer; light chain monomer or dimer; as well as one heavy chain and one light chain; Other dimers are also considered here. This type of antibody fragment is produced by chemical methods. For example, a complete antibody can be treated with a protease such as papain or pepsin. can be made by cutting or e.g. truncated heavy chains and/or produced by recombinant DNA technology using host cells transformed with the light chain gene. You can also Heavy and light chain monomers can be used as well, e.g. dithiothreitol or β-mercaptoethano can be prepared by treatment with a reducing agent such as Created using host cells transformed with DNA that coats the light chain or both. You can also. As an alternative to hybridoma technology, Antibody fragments can also be isolated by phage cloning (e.g. (see Clackson et al., 1991 [16]).

The monoclonal antibody of the present invention has antibody recognition of domain 4 in VCAMI. It can be used for any purpose in which VCAMI and its ligand (VLA -4) includes applications where it is desired to prevent binding between For example, the monochrome of the present invention treatment of inflammation or diseases characterized by the binding of local antibodies to leukocytes to the endothelium; For example, due to trauma after reinfusion, microbial infection or VCAMI-mediated binding pathways. It can be used to treat other cell migration (eg, metastasis), vasculitis, etc.

The monoclonal antibodies of the invention can be combined with other antibodies, bioactive agents, or substances of various interest. They can also be used in combination. For example, the monoclonal antibody of the present invention can be used to treat endothelial cells. 4B9 or other anti-V for the treatment of disorders characterized by VCAM1 expression in cells. Can be used in combination with CAMI antibodies. Or the monoclonal of the present invention antibodies to other endothelial cell receptors identified in inflammatory diseases (e.g. ELAMI, In combination with an antibody that recognizes ICAM1, etc.), we can investigate the scope of the leukocyte-endothelial cell binding pathway. It can also be used in combination therapies designed to suppress. Furthermore, the antiseptic of the present invention or a VCAMI-recognized fragment thereof, such as TNF, ricin or diphtheria. V'CA M1-expressing cells by binding to cytotoxic molecules such as the A chain of toxins. Antibody/toxin conjugates can also be provided that can be directed against cytotoxic elements. difference Furthermore, the antibodies of the invention can be immobilized on a chromatographic substrate (e.g. protein A-Sepharose). Once established, it has priority over VCAM-6D! , :VcAM-7D was separated. Alternatively, affinity chromatographic resins useful for purification can be formed.

The detectably labeled antibodies according to the invention can further be used in vitro or in vivo. for detecting VCAMI protein in samples or VCAMI-expressing endothelial cells. It can also be used in screening or diagnostic methods.

For example, these samples may be tested for the presence of VCAMI or VCAMI-expressing cells. For this purpose, a sample can be subjected to a labeled antibody screening according to the present invention. and whether a VCAMI/anti-VCAMI antibody complex was formed. Detect.

Suitable labels can be radioactive, enzymatic, fluorescent, magnetic or chemiluminescent.

The radiolabeled antibody was prepared using a known method. It is created by combining radioactive isotopes such as IO’), and then it is can be detected by a scintillation counter, a scintillation counter, or by radiophotographic techniques. I can do it. Antibodies of the invention suitably target enzymes such as yeast alcohol dehydrogenase. Label with enzymes such as , horseradish peroxidase, and alkaline phosphatase. can be developed and then detected spectrophotometrically or with the naked eye. Wear. Suitable fluorescent labels are fluorescein isothiocyanate, fluorescamine , rhodamine, etc. Suitable chemiluminescent labels are luminol, imidazole , oxalic acid esters, luciferin, and the like.

For therapeutic use, monoclonal antibodies of the invention may be combined with a pharmaceutically acceptable carrier. It can be formulated as a pharmaceutical composition consisting of effective amounts of the antibodies in admixture. typical For therapeutic use, the antibodies of the invention are suspended in a sterile saline solution for therapeutic use. Or medicine The composition modulates the release or prolongs the presence of the active ingredient in the patient's body. It can also be prescribed to Many suitable drug release systems are known, including hydrogel, hydroxymethylcellulose, microcapsule, liposome , microemulsions, microspheres, etc.

Pharmaceutical compositions contemplated here include, for example, oral, intranasal-subcutaneous, intramuscular, intravenous Administration can be by any suitable means, such as intracorporeally, intraarterially or parenterally. General Intravenous (i,v,) or parenteral administration is usually preferred.

Example I VCAM-7D! ” - I want VCAM-6D! - Shiinai MAb The monoclonal antibody of the present invention that recognizes domain 4 of VCAM-7D was prepared as follows. Created in. Four BALB/C mice were treated with VCAM-6D expressed on their surface. whole CHO cells (“VCAM-6D/CHO” cells) were injected. CHO culture was stably transfected with cDNA encoding VCAM-6D. The hamster ovary cell line was developed by Biogen Inc. Bridge, MA). First, approximately 107 cells were injected into each mouse. Inoculated intraperitoneally (i,p) with BS. Complete Freund's adjuvant in different parts After 2-24 hours of injection (i,p,)L,

These mice were injected intraperitoneally with VCAM-6D/CHO cells without adjuvant. I boosted it twice. Each mouse was then treated with 55 μg of recombinant soluble VCAM- 7D (rsVCAM-7D, N-terminal 674 amino acids of mature VCAM-7D) constructed and lacking transmembrane and cytoplasmic regions; Biogen, Inc. (obtained from Poration, Cambridge, MA) and immunized with incomplete Freund's Injected intraperitoneally with adjuvant. These mice were treated without adjuvant. VCAM-7D (i.

p,) was boosted twice. Four days after injection of rsVcAMl, The spleen was excised from one mouse, and the spleen cells were collected by Lerner [17]. fused with P3/X63-Ag3.653 murine myeloma cells according to the method. Melt Supernatants of individual clonal cultures in combined cells were transferred to rsVCAM-7D-coated plates. was screened for its ability to bind to EL I SA plate (Co sVCAM-7D (0, lμg/m!, 50μl/hole) and incubated overnight at 4°C. Aspirate coating solution and block buffer solution (phosphate buffered saline (PBS) containing 5% fetal bovine serum (Fe3), pH 7.2 ) was added to block the remaining sites and the plate was incubated for 2 hours at room temperature. Ta. Remove the blocking buffer and remove 50 μl of hybridoma culture supernatant (or block buffer). Supernatant (diluted in lock buffer) was added to the plate, then for 1 hour at room temperature. Incubated. The plate was then washed with 0.05% detergent (Tween-20). Unbound antibodies were removed by washing with PBS containing horseradish peroxidase. Incubated with conjugated goat anti-mouse immunoglobulin for 1 hour at room temperature.

Wash the plates with PBS/Tween-20 solution and add chromogenic horseradish peroxide. Sidase substrate, TMB (3,3'.

5.5'-tetramethylbenzidine; ICN, Wrisley, IL) at 42mM Color was developed with 0.1 M sodium acetate-citric acid (pH 4,9). hydrogen peroxide (15 μm 130% solution) and by addition of 2N sulfuric acid. The reaction was stopped. The colored product was transferred to a Termofax plate reader (Model Quantitated at 450 nm using Recura Devices, Inc.; Barco Aldo, CA). .

Cells in positive culture wells were subcloned by limiting dilution, and the above Remeasured by screening analysis. The hybridoma supernatant was then subjected to FAC 3 analysis temporary 1. : Emit VCA M-6D or VCAM-7D. The ability to stain CO37 cells was evaluated. In these experiments, VC An expression vector (pC DM8) and then Ospone et al., 1989 [2] and Hetsion et al. CO37 particles were obtained by electroporation as described in 1991 [9]. cells were transfected.

Transfection with either VCAM-6D or VCAM-7D cDNA The infected CO37 cells were washed and added with suspension buffer (PBS 15mM EDTA/2 %FC310,05% sodium azide), then hybridoma culture The supernatant from the culture was incubated for 45 minutes at 4°C. Then transformer The infected cells were washed with suspension buffer and fluorescein-treated goat anti- -Mouse Ig (Jackson ImmunoResearch 1, West Grove, PA) ) for 45 minutes at 4°C. Wash these cells in PBS. Fixed with 0.2% paraformaldehyde, FAC8tar (Becton Son Inc., Mountain View, Calif.).

FAC3 analysis showed that the isolated anti-VCAMI monoclonal antibody was isolated from VCAM-7. D but not VCAM-6D. Binding of these antibodies The data are summarized in Table 1.

Example 2 Ramos” Ramos binds to VCAMI because it expresses VLA-4, but e.g. Binds to other induced endothelial cell molecules such as CAM-1 or ELAM-1 (Osporn et al., 1989 [2]), a B-lymphoblast cell line that does not Ramos therefore provides a convenient model model for analyzing VLA-4-dependent binding. It is a cell line. Ra　m　s cells are recombinant VCAM-6D and recombinant VCAM- 7D, if these molecules are expressed in CO8 cells. Conventional note As expected, binding to VCAM-7D was determined by a single preparation of each plasmid. slightly more than for VCAM-6D in transient transfection analysis of less than 2 times) larger (Hetsion et al., 1991 [9]). In this binding activity To determine whether quantitative differences are reproducible, several box preparations of each plasmid were prepared. neeloporated into CO8 cells in parallel. Repeat transfection showed that the expression of the two plasmids was similar, but on average V CAM-7D binds significantly more cells per mm than VCAM-6D ( (See Figures 6 and 7). 6-domain and 7-domain types of VCAMI The binding to both anti-VLA-4 MAb HP1/2 CAM MAb 4B9i: more inhibited (Figure 7).

In all these experiments, Ramos cell binding was measured as follows: Target cells (e.g. COS tissue culture plates) in 48-well plastic tissue culture plates. RPMI/10% fetus Wash with bovine serum (Fe2) and add a predetermined amount (e.g. 10 μg/ml, M per experiment). Test MAb (e.g. 4B9, HP 1/2, GH12 ) was added in a volume of 0°15ml. For HUVEC, cells were incubated at 37°C. 20 ng/mI recombinant human TNF (Biogen Inc. 4, 24, 48 or 72 hours The expression of VCAMI was induced by incubation. Ramos cells 2', 7' -bis(2-carboxyethyl)-5(and 6)-carboxy-fluorescein (“BCECF”, Molecular Blobes, Eugene, OR). Fluorescent labeling was performed according to the manufacturer's guidelines. First, centrifuge the cells. isolated and then incubated with 5X10' cells/ml in RPMI/10% fetal bovine serum (Fe2). Resuspend to concentration. BCECF was added to 2 μM and the suspension was incubated at 37°C for 3 Incubated for 0 minutes.

Wash the cells twice with RPMI/10% FC3, then add 3X10' cells to the same solution. /ml.

4. Add 5X10' labeled Ramos cells to each well of a 48-well tissue culture plate. (final volume 0.3 ml) and then incubated for 10 minutes at 37°C. Next The plates were then emptied by inverting them while shaking, and C, a+3 and Mg Washed three times with Hank's Buffered Salt Solution (HBSS) containing ++. Adherent cells 0.1 Dissolved by adding 2 ml of 1% detergent solution (Nonidet P-40) and then Incubate for 5 minutes at 37°C. Multi-channel micropipette lysate Transfer to a 96-well microtiter plate and titrate the number of adherent Ramos cells. Chiku Fluorscan II ELISA Reader (Flo Laboratories) (see Gimbron et al., 1989 [18]).

I don't want to live and die. Antibodies 'i-VCAM-7D, VCAM-6Dt-jJ: and domain 4-dependent Tested for ability to bind to various VCAM/ICAM constructs exhibiting cell binding (See Figures 1-13 and Example 3). Specific VCAM, ICAM (control) or or chimeric vCAM/ICAM (or VCAM/VCAM, see Figure 11). C087 cells transfected with the cDNA construct were transferred to HBSS/ After incubating for 15 minutes at 37°C in 5 m MEDTA, pipette vigorously. The cells were inhaled and removed from the 100ml culture dish.

3-5 x 10' cells were individually incubated with 2 μg/ml of the MAb to be tested. Rabbit polyclones incubated or generated against rsVCAM-7D PNP (PBSlo, 1% sodium solution) together with null antiserum (1:500 dilution) 2% FC3) for 20 minutes at room temperature. incu After baiting, cells were washed twice with PNP, 0.2 μg for 20 min at room temperature. Second fluorescein-treated antibody (rabbit anti-mouse immunization) in PNF / ml globulin or goat anti-rabbit immunoglobulin). . Cells were then washed three times with PBS and suspended in PBS/1% formaldehyde. Store in the dark at 4°C, and use FAC3tar (Becton-Jickinson; Analyzed by Tenview, CA). Produce the relationship between cell number and fluorescence intensity. The number of positive cells in each sample was determined from the FACS histogram.

in parallel for each transfectant and negative control (pCDM8 vector only). Compare the fluorescence peaks obtained and compare these peaks to eliminate 99% of the negative cells. I marked the sea urchin. Count the number of positive cells (to the right of the mark) for each transfectant. It was decided. This method results in a slight underestimation of the number of positive cells in each case. Ta. This is because slightly positive cells that overlap the fluorescence peak of the negative control are eliminated. It is et al.

Example 3 Chimeric VCAM ICAM polypeptide and current To identify the region of VCAMI involved in the recognition of VLA-4-expressing cells, V A recombinant gene encoding a CAM/ICAM chimera was created, which included VCAM- 7D and VCAM-6D cDNA parts were excised and the same Unique restrictions in VCAMI cDNA using similar regions of ICAM-1 Endonuclease site (see Figure 1) and ICAMI polymerase chain reaction A PCR-generated fragment was used. By replacing the truncated VCAMI region, Structural changes associated with deletion of one or more domains in the VCAM1 molecule were eliminated as much as possible. ICAMI was chosen as the source of the substitution construct. Why? This is a member of the 1g superfamily, and the Ig superfamily IJ- This is because it has the highest degree of amino acid homology to VCAMI among its members. (Ospone et al., 1989 [2]).

VCAMI/I Schematic diagrams of CAMI chimeric polypeptides are shown in sections 1 to 5.8.9 and It is shown in Figure 11. cDNA constructs encoding each of these were created as follows. Made: Original Boulasmid VCAMIE II/CDM8 (VCA-M=7D coded) (see Herashun et al., 1991 [9]) *TAi; LVCAM41/CD M8 (codes VCAM-6D; Ospawn et al., 1989 [2]) Each was cut with a restriction endonuclease at the appropriate site shown in Figure 1. , purified on agarose gel. I Live endothelial cDNA with CAMI insert as described above Produced by PCR from the I CAMI/CDM8 plasmid isolated from the library. Ospawn et al., 1989 [2]), which includes the published information of ICAMI. using sequence-based oligonucleotide probes (Simmons et al., 1988 [ 19]; Staunton et al., 1988 [20]). The PCR primers are as follows. Ni designed to Bly7-P-11 (SEQ ID NO: 1) The first 12 nucleotides are VC Corresponding to the coding region of AMI amino acid residues 9-12, the next 23 nucleotides are Corresponds to the coding region of ICAMI amino acid residues 11-18.

Primer P-2 (SEQ ID NO: 2) first 13 nucleotides are VCA Corresponding to the coding region of MI amino acid residues 86-89, the next 21 nucleotides Corresponds to the coding region of ICAMI amino acid residues 84-90.

Primer P-3 (SEQ ID NO: 3) first 12 nucleotides are VCA Corresponding to the coding region of MI amino acid residues −3 to 1, the next 20 nucleotides are I Corresponds to the coding region of CAMI amino acid residues 300-306.

Primer P-4 (SEQ ID NO: 4) first 14 nucleotides are VCA Corresponding to the coding region of MI amino acid residues 92-96, the next 22 nucleotides I Corresponds to the coding region of CAMI amino acid residues 79-85.

Primer P-5 (SEQ ID NO: 5) first 11 nucleotides are VCA Corresponding to the coding region of MI amino acid residues 134-137, the following 24 nucleotides corresponds to the coding region of ICAMI amino acid residues 121-128.

Primer P-6 (SEQ ID NO: 6) first 11 nucleotides are VCA Corresponding to the coding region of MI amino acid residues 302-305, the following 24 nucleotides corresponds to the coding region of ICAMI amino acid residues 279-286.

Primer P-7 (SEQ ID NO near) first 13 nucleotides are VCA Corresponding to the coding region of MI amino acid residues 92-96, the next 18 nucleotides Corresponds to the coding region of VCAMI amino acid residues 385-394.

For the construct VCAM/ICAM-1 (Figure 3), PCR synthesis of the insert was carried out using P-1 and P-1. and P-4, and for the constituents VC, AM/I CAM-2 (Figure 4) was treated with P-1 and P-5; constituents VCAM/I and CAM-3 (Fig. ) was treated with P-2 and P-6; constructs VCAM-6D/VCAM4 -1 (Figure 11) was treated with P-3 and P-7. VCAM-6 D/ICAM-1 and VCAM-6D/I CAM-2 (Fig. 8 and Fig. 9) ), it is the same as (“nVcAM/ICAM-1 and VCAM/ICAM-2 Create a VCAM-7D expression vector (VCAMIE 11/ CDM8) Dena (VCAM-6D expression vector (V CA M 41 / C D M 8) f,:';i Seta to loan. Hession et al. (1991) After PCR synthesis carried out as described in [9], the insert was isolated using suitable restriction vectors. cleaved with endonuclease and gel purified. Expression vector and insert B ligase and buffer, ethanol precipitated, then Biorad Di electroporated into bacterial host strain MC1061/P3 using I let it happen.

Clones were screened by PCR and restriction cleavage, and a small number of Clone at least one isolate using a sequencease kit for conjugates and inserts. (United Stave Biochemical Corporation) The row has been decided. VCAM-7D, VCAM-6D, and each chimeric construct V CAM/ICAM-1, VCAM/I CAM-2, VCAM/ICA M-- 3. VCAM-6D/I CAM-1, VCAM.

Complete cDNA of 6D/I CAM-2 and VCAM-6D/VCAM4-1 The sequences are shown in the sequence list below (sequentially SEQ ID NO: 8-15).

CO37 cells were transfected with the constructs shown, and Well expressed as tested by FAC3 analysis when stained with reclonal antiserum. It turned out that.

Components VCAM/ICAM-1 (Figure 3) and VCAM/I CAM-2 (Figure 3) 4), most of VCAM domain 1 or most of domain 1 and The first half of in2 was replaced by the ICAMI sequence (shaded area, respectively (See Figures 3 and 4). Component VCAM/I CAM-3 (Figure 5) Therefore, VCAM domains 2 and 3 and a small portion of domains 1 and 4 are ICAM I sequence (shaded area, see Figure 5).

Example 4 As shown previously, MAb 4B9 has a statement in VCAMI domain 1. Three domains at the N-terminus of VCAMl support attachment to VLA-4 Teichmann et al., 1991 [211), and anti-VCAMI MAb 4B9+;! , induced HUVEC or VCAMI l-transfect prevents binding of VLA-4-bearing cells to the body (Carlos et al., 1990 [22 ]). These results suggest that as well as being recognized by VLA-4, The binding site of VCAMI that is inhibited by 4B9 is the N-terminal portion of the VCAM1 molecule. exists in

Using the MAb binding assay described above (Example 2), MAb 4B9 It was confirmed that the epitope in 1 was recognized. 4B9 is VCAMI's 7- It binds easily to C087 transfectants expressing both the It was found that it matched. Furthermore, most of domain 1 consists of ICAMI amino acid residues. Chimeric constructs VCAM/I substituted with CAM-1 and VCAM/IC AM-2 (Figures 3 and 4, respectively) did not bind to 4B9 (see Table 1). (see). These results indicate that the epitope bound by 4B9 requires domain 1 for recognition. showed that it was necessary. Finally, the component VCAM/I CAM-3 (Fig. 5, domain 1, almost complete) binds 4B9, and domain 1 contains the 4B9 epitope. It was confirmed that the 4B9 bonding and Ramos block characteristics are shown in Table 1. Summarize.

Constructs VCAM/ICAM-1 and VCAM/ICAM-2 are MAb 4B9 did not bind Ramos cells, but both were found to bind Ramos cells to intact VCAM-7D. The binding was at approximately 30% of the level expected (Figure 7). This combination is VLA-4/VCAM mediated by I. This is because anti-VLA-4 MAb HPI/2 does not interact. Blocking antibodies (MAb) against the ICAMI-1 pathway 60.3) does not affect the binding (data not shown). Constituent VC AM/ICAM-3 bound Ramos cells weakly, but as measured by complete VC It bound at approximately 10% of the level seen with AM-7D (Figure 7).

Two Ramos combinations in VCAMI: VCAM/ICAM -1 and V, Ramos cell binding to CAM/ICA M-2 is domain based on the remainder of the domain 1-dependent binding site or domain 4 (which is composed of domain 1). VCAM/I CAM-1 Create a structure similar to J: and VCA M/ICAM-2, but with the exception of VC AM/fc Copy VCAM-6D (does not include domain 4) instead of AM-7D. The encoding cDNA was used as a substrate or backbone for the substituted I CAMI insert ( (See Figures 8 and 9). These components (VCAM-6D/I CAM-1 and and VCAM-6D/ICAM-2) is an anti-VCAMI polyclonal Although well expressed in CO37 cells as determined by antiserum staining, Ram os cells could not be bound (Figure 10).

These results indicate that the constructs VCAM/ICAM-1 and VCAM/I It seems that the binding of Ramos cells to CAM-2 is based on the presence of domain 4. It will be done.

Next iteration, domain 4 of VCAM-7D will be used instead of domain 1 of VCAM-6D. A construct called VCAM-6D/VCAM4-1 was created (Fig. 11). ).

Referring to the results shown in FIGS. 7 and 12, VCAM-6D/VCAM4- The Ramos binding of 1 is similar to that of VCAM-6D (see Figure 2, complete domain 1) and and VCAM/ICAM-1 (see Figure 3, complete domain 4), Domain 4 is shown to bind cells. This result binds to domain 1 MAb 4B9 does not bind to domain 4 This is somewhat surprising in view of the fact that s-cell binding can be completely blocked. steric obstruction Harm is one possible explanation. Furthermore, the binding of 4B9 to domain 1 is disrupts the structure of the cell, so that domain 4 is no longer in its proper configuration to bind cells. There is a possibility.

Example 5 Monoclonal torsion traces that stop domain 4. According to the procedure shown in Example 1, VCAM-7D is recognized, but VCAM-6D is recognized. We created three types of monoclonal antibodies that do not recognize domain 4 or It was shown to bind main 4-dependent epitopes. These MAbs were GE4,E They are referred to as Dll and GH12. These three MAbs all have IgG1 isotypes. It was a tap.

The binding and Ramos blocking properties of each antibody were evaluated using the above antibodies and Ramos binding properties. The values were determined using a synthetic analysis (Example 2).

MAb GH12 is VCAM-7D, VCAM/I CAM-1 and 2 and l :C087 transfectant expressing VCAMI-6D/VCAM4-1 was bound, but the cells expressing VCAM-6D or VCAM/ICAM-3 The song did not bind to transfectants. Referring to Figure 13, the Ramos combined The experiment was performed using MAb GH12 at 10 μg/ml with VCAM/I CAM-1, VCA CO expressing M/ICAM-2 and VCAM-6D/VCAM4-1 constructs 37) Blocks Ramos binding to transfectants but VCAM/IC It was shown that it did not bind to AM-3-expressing transfectants, indicating that A small portion of domain 4 removed from the CAM/ICAM-3 construct binds GH12. indicates that it is important. MAb G■]12 is VCAM-6D or VC did not block Ramos cell binding to AM-7D.

Binding experiments have shown that MAb EDII is compatible with VCAM-7D, VCAM/ I. Binds to C067 cells expressing CAM-1 and VCAM/ICAM-2. However, to VCAM-6D, VCAM/I CAM-3 or vCAM/VC It did not bind to cells expressing M4-1. With reference to Figure 14, Ramos Binding experiments showed that MAb EDII was conjugated to VCAM/ml at 5 μg/ml. C087 trans expressing ICAM-1 and VCAM/I CAM-2 constructs Although Ramos binding to the sfect body was blocked, VCAM-6D expression and V CAM/ICAM-3 expressing or VCAM-6D/VCAM4-1 expressing ivy that prevents transfectants. Surprisingly, MAb EDll is VCA Partially blocked Ramos cell binding to M-7D-expressing C087 cells .

Binding experiments showed that MAb GE4 is VCAM-7DXVCAM/I Expressing CAM-1, VCAM/jCAM-2 and VCAM/ICAM-3 VCAM-60 or VCAM-6D/VC It did not bind to cells expressing AM4-1. With reference to FIG. s binding experiments showed that MAb GE4 was absorbed into VCAM at 10 μg/ml. C087 transfectant expressing /ICAM-1 and VcAM/I CAM-2 RamO5 binding to the ectoform was blocked. MAb GE4 is VCAM-6D Expresses /VCAM4-1, VCAM/ICAM-3 or VCAM-6D did not prevent Ramos attachment to transfectants and were further tested. Also blocked Ramos binding to VCAM-7D-expressing COS7 cells at concentrations There wasn't.

Binding and inhibition data are summarized in Table 1. From these results we can conclude Each of the three domain 4 antibodies recognizes a different epitope, two of which CAM-1i: capable of blocking domain 4-dependent attachment of VLA-4 to Wear. These antibodies are a new class of anti-VCAMI blocking antibodies.

Table 1 Component 1 MAb VCAM-7D VCAM-6D A B CDH12 Connectivity + −+ 10−+ Blocking property + +-+ D11 Connectivity + −++−− Interdiction +/-+ +-- Connectivity 10 −＋＋＋− Interdiction - 1111 4B9 (control) Connectivity + +−−+− Inhibition + +--+- *A=VCAM/ICAM-1 B=VCAM/ICAM-2 C=VCAM/ICAM-3 D=VCAM-6D/VCAM4-1 Example 6 Written by Cytokine Activated Ramos domain 4 MAb stopping domain-4-dependent MAb, EDI I or GH12 is VCAMI in endothelial cells (this is mainly VCAM -7D) in humans to determine whether it is possible to prevent adhesion to Vascular endothelial cells (HUVEC) were treated with 20 ng/ml human recombinant TNF for 4 hours. Ram, treated for 24, 48 or 72 hours.

The binding of S cells and the ability of antibodies to block binding was determined. All MAbs are 10μ It was added at a concentration of g/ml. The results are shown in Figures 16 to 19 for each activation time. show.

Overall, these results demonstrate that MAb GH12 was at a single concentration tested (10 μg /ml) does not reliably block HUVEC/Ra m o s binding, but MAb EDII is Ram for induced HUVEC for all times tested.

This shows that more than 80% of S cell binding was inhibited. MAb4B9 is more than 90% H UVEC/Ramos binding was blocked. As these results suggest, VCA The two cell binding sites of MI (domain 1- and domain 4-dependent) are separated. They retain functionality with each other, and antibodies against each are Ramos antibodies against the other. Can interfere with cell binding. This is true even though antibodies do not cross-react with each other's binding sites. (as shown by the antibody binding data for the above constructs).

As in this example, VCAM-7D is primarily responsible for the expression of VCAM-1 (RNA and and stimulated endothelial cells, whereas VCAM-6D ( RNA) is the main VCAM in bone marrow stromal cells and tonsil follicular process cells. -1 type. This indicates that the domain 4-specific anti-VCAMI antibody The present invention suggests therapeutic advantages over specific anti-VCAMI antibodies. domain 4 antibody strongly inhibits the recruitment of leukocytes to the site of inflammation via stimulated endothelial cells. This suggests that VCAM-1 does not block lymphocyte formation, suggesting that VCAM-1 does not inhibit lymphocyte formation. (Myake et al., 1977 [7]). This type of B-cell Blocking lymphopoiesis may be undesirable during long-term treatment with anti-VCAMI antibodies. stomach.

Although the present invention has been described above with reference to Examples, the present invention is not limited to these Examples. It will be readily apparent to those skilled in the art that many modifications may be made from these disclosures. cormorant.

Therefore, all these modifications are also within the scope of this invention.

Under the Budapest Treaty, the applicant has provided hybridoma-producing EDII, GE4 and GH12 monoclonal antibody subclones were cultured in American type culture. ・Collection (ATCC) (12301 Park Lawn Dr, Otsuk Building, M D (US)) on January 9, 1992. Cultures were identified as follows. Caught: Ha ATCC collapse mouth MAb CB, EDIl, AC3HB 10962 MAb CB, GE4. B G5 HB 1091096l CB, GH12, AA12 HB 10963 Cited publications The above publications are incorporated herein by reference.

array list (1) General information: (i) Applicant: Osbon, Laurelly Benajamin, Christopher D.

(ii) Name of the invention: Recognizing the fourth immunoglobulin-like domain of VCAMI antibodies (i　i　i) Number of arrays: 23 (iv) Correspondence address: (A) Address: Allegretti & Witkots, Limited (B) Town name: South Waraker Drive No. 10 (C) Car name Nijikago (D) State name: Illinois (E) Country name: U, s, A.

(F) Z I P: 60606 (V) Computer reading type: (A) Media: Floppy disk (B) Computer: IBM PC compatible (C) Operation system: PC- DO8/MS-(D) Soft Air: Patent in Release No. 1.0 , Version No. 1.25 (vi) Current application data (A) Application number: CB) Application date 2 (C) Classification: (viii) Agent information: (A) Name: Matsuku Nicholas, Janet M9 (B) Registration number: 32,918 (C) Reference number: 92,306-A; DOOI CIP PCT (ix) Telegraphic information: (A) Telephone: (312)-715-1000 (B) Telefax: (2)-8EQ ID NO: 1 (7) Information: (i) Array characteristics: (A) Length = 35 base pairs (B) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology bilinear (xi) Array (7) Explanation: SEQ ID NO: 1: ℃ x heart low = Ni = Ri Cα mouth View 1 (2) Information on SEQ ID NO:2: (i) Sequence characteristics: (A) Length = 34 base pairs CB) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology bilinear (xi) Sequence explanation: SEQ ID NO: 2: α=αFlo M℃fudχ^KoniC (X;Al鄭ΩχπαmaA(2)SEQ ID NO:377) Information: (i) Distribution Column characteristics: (A) Length: 32 base pairs (B) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology bilinear (xi) Explanation of array: SEQ ID NO: 3: ffi Ryo = Fugo τ press χ lever = ω (2) Information on SEQ ID NO: 4: (i) Sequence characteristics: (A) Length = 36 base pairs (B) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology: Linear (xi) Sequence description: SEQ ID NO: 4: α gift m - pigeon χ = χyu χ punishment bias height (2) Information on SEQ ID NO:5: (i) Sequence characteristics: (Δ) Length: 35 base pairs (B) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology bilinear (xi) Sequence explanation: SEQ ID NO: 5: W co’IITGA n 2 A CCAC (2) SEQ ID NO: 6 information: (i) Sequence characteristics: (A) Length: 35 base pairs (B) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology bilinear (xi) Array description: SEQ ID NO: 6: ωbuχ loco pressure πz side C: χ Terue (2) SEQ ID NO Near information: (i) Sequence characteristics: (A) Length: 31 base pairs (B) type: Nucleic acid (C) Mirror-shaped knee heavy chain (D) Topology: Linear (xi) Sequence description: SEQ ID NO: 7: Kuchikara = = Hato Konsho ni Jochi χWC (2) Information on SEQ ID NO:8: (i) Sequence characteristics: (A) Length: 2217 base pairs (B) type: Nucleic acid (C) Mirror type: double strand (D) Topology: Linear (ix) Features: (A) Name/Symbol: CD5 (B) Position: 1. ．． 2217 (1x) Features: (A) Name/symbol: m'at peptide (B) Position near 3. ．． 2217 (ix) Features: (A) Name/symbol: sig peptide (B) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 8: (2), SEQ ID NO: 9 Information: (i) Sequence characteristics: (A) Length near 39 amino acids (B) type diamino acid (D) Topology: Linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 9: Asn Ala Thr La u Thr IJII Engineering 1a Ala Ma se Arq Mat Glu As p Sar Gly 1eum Trp Sir Arg Gin um P ro Asn Gly Glu M loss G’in Pro h Ser Glu ( 2) Information on SEQ ID NO:10.

(1) Sequence characteristics: (A) Length: 1941 base pairs (B) type: Nucleic acid (C) Mirror type: double strand (D) Topology bilinear (ix) Features: (A) Name/Symbol: CD5 (B) Position: 1. ．． 1941 (ix) Features: (A) Name/symbol: mat peptide (B) Position 73. ．． 1941 (ix) Features: (A) Name/symbol: sig peptide (B) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 10: AGA AAG GAA ω ℃GAA TTA ATT ATCCAA Grr ACI’α λIiA G ACATA AA included 1536CTr ACA GCT TIT (IT'I 'CrGAP AGT GICMA GAA GGA GACACr G'll CATC1T84 ATCTCr! ACA TGr GGA MT GrT CCA GAA ACA TGIII; jlfl ATCG℃AAG 1GR2 TMAce A! αs NCα:c (%’rrc AAG wαko αa c’ n’w GAAπ7 1728 prisoner hum c’rrrm s MAG (engineering AAC ℃N GαηΔko, πaτaλ 1920 (2) SEQ ID NO: 11 information Information: (i) Sequence characteristics: (A) Length = 647 amino acids (B) type diamino acid (D) Topology: Linear (ii) Molecular type: protein (xi) Array description: SE’Q ID NO: 11: Val Ltax Tyr Pha Ala Sar Ser 1 engineering 1e engineering le Pro Ala engineering 1e Gly Mat engineering 1e engineering le last 1 glaze Ala Ar+g Lys Ala Ag n Met Lys Gly Ser Tyr Sar Lau Va1 (2) Information on SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 2205 base pairs (B) type: Nucleic acid (C) Mirror type: double strand (D) Topology bilinear (ix) Features: (A) Name/Symbol/CD5 (B) Position: 1. ．． 2205 (ix) Features: (A) Name/symbol: ma t peptide (B) Position: 73. ．． 2205 (ix) Features: (A) Name/symbol: sig peptide (B) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 12: ACII: TCCTGI' GACCAG Q! AAG TrG 'I'm℃GGCATA GAG A Engineering rate α 192MA AAG GAG’rrc arc C1℃COT G GG MCMCαnAAGGTG’1M’GMCI11m 240AGCAAr GrGGAGAAGATAGCGACCAAffGTGCTrTCAMCTGC CQ' 288 (2) SEQ ID NO: 13 information: (i) Sequence characteristics: (A) Length, 735 amino acids (B) type diamino acid (D) Topology/linearity (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 13: Ala Lys Leu H is　Ile　Asp　Asp　Mat　lu　F’na　Glu　P! ℃L ys Gin Arg G1n (2) SEQ ID NO:14 information: (i) Array characteristics: (A) Length: 2208 base pairs (B) type/nucleic acid (C) Mirror type: double strand (D) Topology bilinear (ix) Features: (A) Name/Symbol/CD5 (B) Position: 1. ．． 2208 (ix) Features: (A) Name/symbol: mat peptide (B) Position near 3. ．． 2208 (ix) Features: (A) Name/symbol 2sig peptide (B) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 14: GAA (I; G c’rG GAA crGG(S αI: CIC(ICTCTTGG QliGαλGrG GGCAAG 384CK: AcCcrGGIIG C1℃CTCCmGGG tic AAGCrCkrG AN; XπCAGGM 480 (TAACCT TTACTrGrCjflGAGGATATl”GGAAAAGn’CrrGr rTGC576GAG GCr (n AAA GAA TTG OIA c’ rc'x to s'nsu α): MGAATACA engineering 672 (2) SEQ ID Information on NO: 15: (i) Sequence characteristics: (A) Length near 36 amino acids (B) type diamino acid (D) Topology: Linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 15: (2) SEQ ID NO: 16 Information: (i) Sequence characteristics: (A) Length: 2220 base pairs (B) type: Nucleic acid (C) 1mm type double strand (D) Topolon - bilinear (ix) Features: (A) Name/Symbol: CD5 (B) Position: 1. ．． 2220 (ix) Features: (A) Name/symbol: ma t peptide (B) Position near 3. ．． 2220 (xi) Features: (A) Name/symbol/sig peptide (B) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 16:℃απαz MG mc 'h-ATCC] Z α included Crr GCr CXI; ATT GGr GAC’I’m GITI: 'n TrG ACT TGCAGCP44 CTGCX;ACAGIGAQ:ATCTX:AGCTTr(II;GQ;QI GAGAffCTC! :CCr　960CCCAAA　CAA　AGG　(X; ACr A(I; CAA ACA CTT T! GrCAAT Grr Go: CC! :　P536 fl GAITACA A’s GrC’ff; GrCAGCQITTmTCCAT CCTGGAGGAAGGC1584 (2) Information on SEQ ID NO:17.

(i) Sequence characteristics: CA) Length near 40 amino acids (B) type diamino acid (D) Topology; linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 17:'Li;ru Asp Ij uArg Pro Gin Gly Lau Glu Shishi Fha Glu A sn Thr Sar ALa17Q 175 180 Sar Sar Val Asn Met Thr Cys’Lsa Sar Gin Gly’F’r＞e Pro Ala Pro@Lys Pha Pro Ser Glu Sar Val Lys Glu Gly Asp Thr Val engineering 1e engineering la Sar Cys (2) SEQ ID N Information on O:18: (i) Sequence characteristics: (A) Length: 1929 base pairs (B) type: Nucleic acid (C) Mirror type: double strand (D) Topology: Linear (ix) Features: (A) Name/Symbol: CD5 (B) Position: 1. ．． 1929 (ix) Features: (A) Name/symbol: mat peptide (B) Position near 3. ．． 1929 (ix) Features.

(A) Name/symbol: sig peptide CB) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 18: CrGGAG GAT GC A GACXCAAG '1m COG G AACCAJlill; AGI 'TTGGAAe　528AQ:TTTACTa:rGTCATTGAGGAT ATrGGAMAG’1TCrTGTf’TGCQlhama 576α:T MA TryNOIICm car GAA x z’rcr c’rcα:CAC A GIA stone CAG 624α:'r c'n AM GAA c' rc'x し'rcxα)l:AAGjlArAcAGrrATr 672T crGTII, AATCCATCCACJMGCTGOuGAAQlrGGCT CT (mAc!: ATI: 720Ace TGr TCr: AGCQ%G G Gr　CAROn　GCI’　CCA　GAG　ATr　’πA to ITCTGG AG　768AGT　rrc　GAA　m　ACC’ri℃ATCCCT　A G: ATr GAA (aT ACTαaAMGcr 1Li2CTG τG ICAGCAGG(%CTCCCTAACGGGGAGCTACI’1G(rr cTTTcrGAG 1392α KTGAGχπαMAGJ α end GAC π Engineering ATCx T (GoππAα 1584GM AACMCAAA GAC'f fl　Trr　’KT　Q? r　C,<　CTT　CrCG11lu　&’a T TTT 1824 GO″ro: π℃ to λ to α℃ to α℃ to ℃ to Su zuχηzu αλ 1872:, A MA GCCAACAm AAG GGG TCA TM XiT C1τc’nmα: ACAGAM 1920 Prize AAA c’rc Ig2g Sar　Lys　Val (2) Information on SEQ ID NO: 19: (i) Sequence characteristics: (A) Length: 643 amino acids (B) type diamino acid (D) Topology: Linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 19: Mat F’ha Ala　ALa　Sar　Gin　Ala　Pba　Lys 1a　Glu　 Ihr Thr Pro Gl■ Lys Ala ■uRetsuLys Asp Ala GIY Vll Tyr G lu ays Glu Sir Lys AmSar Lys Val (2) Information on SEQ ID NO: 20: (i) Sequence characteristics: (A) Length: 1932 base pairs (B) type: Nucleic acid (C) Mirror type: double strand (D) Topology bilinear (ix) Features: (A) Name/Symbol: CD5 (B) Position: 1. ．． 1932 (ix) Features: (A) Name/symbol: mat peptide (B) Position +73. ．． 1932 (ix) Features: (A) Name/symbol: sig peptide (B) Position: 1. ．． 72 (xi) Sequence explanation: SEQ ID NO: 20: ℃Mαa℃a included ^Q4; CCM C! l171tMG TGC flow πλAACπ℃αko 288MA T CA AM GrG 19:12 (2) SEQ ID NO:21 information: (i ) Array characteristics: (A) Length 2644 amino acids (B) type diamino acid (D) Topology bilinear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 21: Lys Sar Sense Glu Mat Thr Fhe Ila Pro’Ihr 1e Glu Asp Thr Gly Lys 45 350 55 360 Val Glu Lal11a Engineering Is Gin Val’Ihr Pro Lys Asp engineer L voice mushi’Ihr Ala (2) SEQ ID NO: 22 Information: (i) Sequence characteristics: (A) Length: 1941 base pairs (B) type: Nucleic acid (C) Mirror type: double strand (D) Topology bilinear (ix) Special @: (A) Name/Symbol: CD5 (B) Position: 1. ．． 1941 (ix) Features.

(A) Name/symbol: mat peptide (B) Position +73. ．． 1941 (ix) Features: (A) Name/symbol: sig peptide (B) Position: 1. ．． 72 (xi) Sequence description: SEQ ID NO: 22: AGTTTGGAAGTAA Q:TTACTO! &ATrGAG(aTATI’GGAAAAGrr 57 6 (2) SEQ ID NO:23 information: (i) Sequence characteristics: (A) Length 2647 amino acids (B) type diamino acid (D) Topology bilinear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 23: Met Pro Gly L ys MgtVal Val Engineering 1a Mushi Gly Ala Sar Asn Engineering e Iau Trp 1a Mat Pha Ala Ala Ser Gin La fha 'Ihr Val Glu Engineering 1a 5er Pro GlyP roArg engineering la Jua Ala Gin engineering le Gly A1%l Sar Val Mat 1 total 9 si “Val Met Gly Cys Glu S ar　Pro　Sar　Ihe　Sir　Trp　Arg　’Ihr　G’ln Work Iek Ya 25 30 Ko 5 40 Ser Gin Glu Fha LeuGlu Asp La Asp Ar g Lys Sar Mushi Glu’Ihr LysSar Ij! uGlu Val　Ihr　! 'ha'Ihr Pro Val Engineering 1e Glu As p engineering le Gly Lys Va■ Li5 160 165 rAVal Cys Arg Ala-Si vHis Engineering 1a Asp Glu M at Asp Ser Val Pr.

170 175 1JO Mn ThrVal Engineering 1a Ser Val Asn Pro Sir’Ih r Lys Mushi Gin Glu Gly GlyFhe Trp 5er L ys Lys Leu Asp Asn Gly Asn Shida Gin His 　Saar　GlyAsn　Ala’Ihr　Li1y　Thr　Leu 1 e, a Met Arg Mat Glu Asp Ser Gly l1e Sar Val Tyr Pro IjuAsp Arg Lsu Glu Engineering 1e Glu Ltm b Lys Gly Glu Keiko 1e Lau Glu Asn Engineering 1e Glu F’ha Xtsx Glu Asp Thr As p Met Lys Ser 30 3 5 340 Leas Glu Asn Lys Ser Lama Glu Met’I hr Fha Engineering 1e Pro 'Ihr Engineering 1e Glu A Tang ■ Fig, 6 Positive cells per io, ooo Fig, 7 Fig, 10 Combined Ramos cells/mm Fig, 12 Combined Ramos cells/mm Fig, 13 Combined Ramos cells/mm Fig, 14 Fig, 15 Combined Ram os cells/mm Fig, 16 Combined Ram os cells/mm Combined Ram os cells/mm Fig, 18 Combined Ramos cells/mm Combined Ramos cells/mm International search report PCT/US 931000311m-km-Nm ρCT/u s　93100031-　PCT/US　93100031 Continuation of front page (51) Int, C1,6 identification symbol Internal office reference number C07K 14152 14/78 8318-4H C12N 5/10 Cl3F 21102 ZNA C9282-4BGOIN 331577 Z 7055-2J//C12N 15102 (C12P 21108 CI2R1:91) (C12P 21102 CI2R1:91) 9281-4B I C12N 15100C

Claims (15)

[Claims] 1. An epitope dependent on the fourth immunoglobulin-like domain of VCAM-7D was identified. A monoclonal antibody that recognizes 2. Blocks Ramos attachment to VCAM1-expressing cells, but monoclonal The monoclonal antibody according to claim 1, which does not cross-block local antibody 4B9. . 3. The following groups produced by hybridomas: (a) ED11. AG6 (ATC CHB10962), (b-) GE4. BG5 (ATCCH-B10961), and (c) GH12. AA12 (ATCCHB10963) The monoclonal antibody according to claim 1, which is selected from the following. 4. ED11, Fab, Fab', F(ab)2 and F(V) fragments, then The derived chimeric antibody, the heavy chain monomer or dimer produced therefrom, and the light chain monomers or dimers produced therefrom, or 1 produced therefrom; A monoclonal antibody with dimeric characteristics consisting of 2 heavy chains and 1 light chain. 5. GE4, Fab, Fab', F(ab)2 and F(V) fragments, then induced chimeric antibodies derived therefrom, heavy chain monomers or dimers produced therefrom, or one light chain monomer or dimer produced from A monoclonal antibody with dimeric characteristics consisting of a heavy chain and one light chain. 6. GH12, Fab, Fab', F(ab)2 and F(V) fragments, and The derived chimeric antibody, the heavy chain monomer or dimer produced therefrom, and the light chain monomers or dimers produced therefrom, or 1 produced therefrom; A monoclonal antibody with dimeric characteristics consisting of 2 heavy chains and 1 light chain. 7. Binds VCAM-7D but not VCAM-6D, VLA-4 expression the binding between sex cells and VCAM1-expressing cells at least partially; A monoclonal antibody that does not cross-block anti-VCAM1-monoclonal antibody 4B9. null antibody. 8. Claim selected from monoclonal antibody ED11, GE4 or GH12 The monoclonal antibody according to item 7. 9. (a) ED11. AG6 (ATCCHB10962), (b) GE4. BG5 (ATCCHB 10961), and (c) GH12. AA12 (ATCCHB10963) A hybridoma selected from the group consisting of: 10. When purifying VCAM-7D from a solution that also contains VCAM-6D, The solution is brought into contact with a substrate on which the monoclonal antibody according to claim 1 is immobilized. A method for purifying VCAM-7D, characterized by: 11. Separation of cells expressing VCAM-7D from cells expressing VCAM-6D In this case, a solution containing cells expressing VCAM-7D is treated with the anti-inflammatory agent according to claim 1. expressing VCAM-7D characterized in that the body is brought into contact with an immobilized substrate. Cell isolation method. 12. VCAM-7D or VCAM- in biological samples or mammals In detecting the presence of cells expressing 7D, the biological sample is as defined in claim 1. In vitro or is an in vivo detection method. 13. Mammalian cell-cell adhesion mediated by VCAM-7D In treating a disease, a mammal afflicted with said disease is given a VCAM-7D-mediated an amount effective to at least partially inhibit cell-cell adhesion of VC in vivo. AM1 domain 4 monoclonal antibody or its Fab, Fab', F(ab) 2 and F(V) fragments, chimeric antibodies derived therefrom, and heavy antibodies produced therefrom. chain monomer or dimer, light chain monomer or dimer produced therefrom or a dimer consisting of one heavy chain and one light chain produced therefrom. A method for treating the above-mentioned disease, characterized in that: 14. ED11. AG6 (ATCCHB10962), GE4. BG5 (ATC CHB10961) or GH12. AA12 (ATCCHB10963), or Fab, Fab', F(ab)2 and F(V) fragments thereof, derived therefrom. chimeric antibodies, heavy chain monomers or dimers produced therefrom, or or one light chain monomer or dimer produced therefrom. Monoclonal antibodies selected from dimers consisting of a heavy chain and one light chain are used as pharmaceuticals. A therapeutic reagent comprising an acceptable carrier as described above. 15. (1) A test selected from ED11, GE4 or GH12 as a reagent. releasably labeled monoclonal antibodies; their Fab, Fab', F(ab)2 also or F(V) fragments; chimeric antibodies derived therefrom; heavy chains produced therefrom; Monomer or dimer; light chain monomer or dimer produced therefrom ; or a dimer consisting of one heavy chain and one light chain produced therefrom; and and (2) complete instructions for using said reagent according to the method of claim 12. A diagnostic kit.