JPH03500659A - protein micelles - 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] protein micelles [Background of the invention] The present invention relates to adhesive proteins (defined below), and in particular to monocytes, granulocytes, and cells. Most of the cells include cytotoxic T lymphocytes, B lymphoblastoid cells, platelets and fibroblasts. Cylindrical function, a glycoprotein widely distributed on the surface of all human cells Regarding associated antigen-B (LFA 3).

LFA-3 is another glycoprotein observed on the surface of T lymphocytes, the differentiation barrier. It is the ligand of the raster second component (CD2).

These two glycoproteins interact and promote adhesion of T lymphocytes on target cells. mediate Similarly, the attachment of thymic lymphocytes to thymic epithelial cells D2 and thymic epithelial cells, FA-3, require 41 points. refined form LFA-3 inhibits the cell-to-cell adhesion of T lymphocytes and red cells, and Mediate aggregation.

[Disclosure of the invention]

The present invention provides micelles of adhesive proteins (e.g. LFA-3), which Naturally contains a fatidylinositol lipid (“PI”) anchor; (preferably containing less than about 10 molecules of protein) is the majority on the cell surface. can bind to target molecules in a multivalent manner. Therefore, the present invention provides the K of LFA-3 to cells. CD such that d is lower than the Kd of monomeric LFA-3, which has been determined to be approximately 400 nM. The present invention provides purified LFA-3, a multimer with binding activity to -2-containing cells. Than Preferably the Kd is -50 nM, even more preferably <20 nM. child Trowel Adhesive Tongue 2 Defined as any protein that mediates force and forces on the surface of a cell. It is a protein present on the top. Here, the target molecule is an adhesive protein that selectively binds (i.e., binds more strongly than any other molecule would be bound and is suitable for is defined as a molecule such that the bond is non-alternative).

The micelles of the present invention have a first cell on its surface and a second cell on its surface in many forms. If the second cell of the first cell has the PI anchor transport protein capacity that can bind to the molecule, It can be used to inhibit cell adhesion. The method is to use a second cell and a second cell. , preferably less than about 10 such proteins, capable of binding multivalently to a large number of molecules, preferably less than about 10. contacting micelles (or micelle-forming, binding fragments thereof) of the molecules. Ru.

The adhesive protein micelles of the present invention can also be used to treat other cells of the patient's activated T cells. physiologically to achieve a bloodstream concentration of LFA-3 effective to inhibit binding to overdose by administering LFA-3 micelles in a consistent buffer to the patient. can be used to treat medical conditions characterized by the presence of activated T cells in : The resulting bloodstream concentration of LFA-3 is preferably between 0.4 and 4.40 nM. It is LF A-3. Excessive activated TM! Characterized by the presence of 3 vacuoles Disease conditions that occur include multiple sclerosis, sarcoidosis, juvenile diabetes mellitus, and systemic Erythematous lupus, thyroiditis, rheumatoid arthritis, ankylosing spondylitis, primary biliary terminal cirrhosis , autoimmune hemolytic anemia, immune thrombocytopenia @ plaque disease, electromyography, allograft Includes single-sided rejection and graft-to-host disease.

Another aspect of the invention is the binding of any adhesive protein that naturally contains a PI anchor. It features a method to increase activity and decrease dissociation constant. Here surfactant An agent is a chemical compound that has hydrophobic and hydrophilic regions that are spatially separated throughout the molecule. defined as a small amphipathic molecule (i.e., one end is charged or polar). and the other end is nonpolar). Critical micelle concentration (CMC) is the concentration of micelle in solution. It is the concentration of a particular surfactant in solution at which cells just begin to form.

This CMC is different for different surfactants.

A further aspect of the invention provides that peripheral blood mononuclear cells or T lymphocytes are treated with antibodies against CD2. By contacting the body with LFA-3 micelles, these cells can be activated or increased. It features methods that have an effect on reproduction.

[Best mode for carrying out the invention]

First, the diagram will be briefly explained.

Ward FIG. 1 is a graph showing the results of two types of gel filtration of LFA-3; Figure 2 is a schematic diagram of the transmembrane (TM) type of LF A-3; Figure 3 is a schematic representation of adhesive protein micelles; Figure 4 is a diagram of human TMLFA -3c　D　NA　A DNA sequence and deduced amino acid sequence; Figure 5 is a graph of the time course of dissociation of LFA-3 micelles from Jurkat cells. the law of nature; Figures 68 and 61] show LFA-3 on Jurkat and resting T cells, respectively. The same data analyzed by equilibrium binding of micelles and the Scatchard method are shown. Figures 7a and 7b are graphs of LFA-3 and CD2 monochrome. local antibody (MA+)--showing time-course and dose-responsiveness of induced proliferation. The graph is: Figure 8 shows Jurkat cells induced by LFA-3 and CD2 MAb. This is a graph of the emission spectrum showing the cytoplasmic fluidity of Ca+2 with time in the cell. the law of nature; Figure 9. Time in resting T cells induced by LFA, -3 and CD2 MAb. It is a graph of the emission spectrum showing the cytoplasmic fluidity of C a+2 according to; Figures 10a and 10b are a set of emission spectrum graphs and histograms, respectively. Therefore, the concentration dependence of Ca+2 influx is due to the LFA-3 occupancy of CD2 in gel-cut cells. This shows that the number of people living in Japan is increasing.

Dan Enobi once! 111 The following cell lines were used in the study of the present invention. Peripheral blood mononuclear cells (PBMC) are Strang sedimentation and Ficoll-Heiberg (Sigma, St. Louis, MO) density It was isolated by centrifugation. Peripheral blood T lymphocytes (PBL, -T) are Wool (Polyscience, Washington, PA) for filtration and plastic adhesion more concentrated. Jurkat cell line was prepared by Dr. M, K, Ho (DuPont, Obtained from Boston, MA). Cells are RPM l-1640゜10% maggots Child serum (Gibco, Grand Island, NY) 5 IIIM gluta min, 50 μg/wl gentamicin (complete medium). LFA-3 and JYB lymphoblastoid cells and Jurkat cells for CD2 purification were MI Expanded at the T Cell Culture Center (Cambridge, MA).

LFA-3 is a human red blood cell (Dustin, M.L., et al., Journal of Experiments). Li, Mental Medicine, 165:677 (1987)) or J’l’B Lin. Pablastic cells (Waller, B. P., et al. Journal of Experimental Medicine) 166:923 (1987)), or Sphatidylinositol-specific phospholipase C (PTPLc) treated J’r 'B lymphoblast cell supernatant ia;-, TS 2. /9 M A b-ce 7' D-S CL 4 B (77 Lumacia, Hiscataway, NJ) on Purified by immunoaffinity chromatography. The latter! 4, 50 Two live J) cells were washed with Hank's Buffered Salt Solution (HBSS) and the total volume Incubate Bacillus thuringis for 1 hour at 37°C at 100y1. rin　1ensis) P　I　P　LC (from Dr. Martin Lo-) Processed at London University, NY, NY). The enzyme concentration used was 0.1% deoxidized 300/nm, ol/llin/n at pH 7 in the presence of sicolate surfactant. 3) (-Phosphatidylinositol 3-hydrolysis is not possible (Lo Ji, M.G., Med. Sods in Enzymology, 71 near 41 (1981)).

Cells were treated with 1100Oxy and cell debris was removed with 100,0000xy for 1 hour. Turn into a beret. Affinity color the lysate or supernatant containing LFA-3. Wash the column by passing it through the column (Dust in, M).

L. et al., supra). LFA-3 from red blood cells is 0.1°〈octyl glucoside ( OG) Eluted from immune affinity carano at pH 3 in the presence of surfactant.

, LFA3 from PIPLC supernatant was eluted at pH 3 in the absence of surfactant, The fractions containing LFA-3 were pooled (46al), and the fractions containing 1 and 1 were equilibrated with TSA. Pass through a phenyl-Sepharose column (Pharmacia). Some prepared samples are MAPS binding buffer and LFA-3 from erythrocytes (with 1/6 OG) Iorado, Richmond, CA) diluted 1:1? ! ? Az1 protein The mixture was passed through a Sepharose CL-4B column.

LFI-3 purified as described above has two forms: red blood cell or MLFA type 3 isolated from lymphoblasts, and MLFA type 5 isolated from lymphoblasts. LFA type 3. mLFA-3 is an intact phosphatidylinositol (P■) anion. It is also called a lipid-bound type. 5LFA 3 is PTPLC' It had a severed PI membrane anchor. Of these two, only mLFA-3 Can form micelles.

A third type called transmembrane LFA-3 (or TM LFA-3) There is LFA-3, which does not have a lipid membrane anchor but is completely LFA-3. It remains attached to the cell membrane by the transmembrane region, which is a part of the 3 protein. ing.

Like mLFA-3, TMLFA-3 can also form micelles, which are described in more detail below. It will be. First, 5LFA 3 will be explained.

Approximately half of FA-3 on the surface of B lymphoblastoid cells was cleaved by PTPLC. Our recent finding that PT is attached to membranes by a cleavable PT glycan moiety (D Ustin JLL, Rane Shah, Hamadan: 546 (1987)). It is possible to obtain a non-aggregated form of LFA-3 (i.e., without micelle formation) for It's now possible.

LFA-3 released from the surface of living JY cells by PIPLC is immunoaffinity chromatography, hydrophobic on phenyl-sepharose Interaction chromatography and protein A affinity chromatography Refined by fee. The middle step removes traces of membrane material or released during enzymatic treatment. This was added to remove LFA-3 micelles that would otherwise be present.

The yield from 50 g of cells was approximately 100 μl. This substance is N-glycanase 25. corresponding to lipid-bound forms from JY cells and erythrocytes when treated with. A single band of 5KD was obtained.

The structure of mLFA=3 in the form of micelles as used in the activation experiments below is shown on the gel. Researched by Sawagaru. The results are shown graphically in FIG. In the presence of 1% OG and The size of mLFA-3 in the absence of bacteria was tested by HPLC gel filtration. interface In the absence of activator (solid line), TSK G54000 or Solpax GF250 ( DuPont) column, IIILFA-3 is thyroglobulin (600,0OOK) D) Eluted slightly before but after blue dextran, 700 KD globular It had a diffusion coefficient similar to that of proteins. The dotted line represents the elution profile in the presence of 1% OG. It shows the feel. In the presence of 1% OG, all LFA-3 is converted to TgG ( Co-eluted with 150K D).

LF A-3 moves to 55-70KD in 5DS-PAGE (Dus tin, M. L., et al., supra). mLFA in the presence and absence of surfactant The molecular weight of 3 cannot be estimated without sedimentation data; however, these data +n about 5 monomeric or dimeric protein micelles in the absence of detergent. This shows that LFA 3 is formed. Based on 5DS-PAGE, + nLFA 3 does not form a disulfide-linked dimer, but a non-covalent dimer. formation cannot be excluded. Furthermore, in the absence of surfactant, sLFA-3 is f with a diffusion coefficient matching that of a mer or dimer: exists as a soluble molecule .

The second type of micelle-forming I-F A-3 is the transmembrane type <TllL FA-3). , the mRNA encoding the second type is mT, FAAB It is different from the type +n R, N A, but only the transmembrane and Only the cytoplasmic region. CD2 binding domain (extracellular part) of both types of LFA-3 are the same.

TM LFA-3 does not have a lipid anchor, but the cytoplasmic region contains hydrophobic amino acids. It contains an acid sequence and therefore remains bound to the purified protein. Micellar shape protein-protein interaction rather than lipid-lipid interaction. Although the resulting micelle formation is the same. TMLFA-3 and +nLFA3 micelles have comparable stability and target binding activity .

The TM form of LFA-3 is naturally present in most aggregated cells and is often It has been observed that lipid-bound forms are also often produced simultaneously.

Mutual jJjIe Micelles of mLFA-3 and TMLFA-3 were prepared using a Centricon 30 device (Amicon , Danvers, MA), in each cycle 21! l of phosphoric acid Three cycles of ultrafiltration adding strong salt solution (PBS) and reducing the volume to 50 μl. Prepare by filtration. The f+terminal P solution in the step after R is the same low fraction as the final retentate. Although it contains molecular components, it does not contain LFA-3, so this ultrafiltration is as follows. It is used as a control to examine the effects of buffer components in experiments.

Formation of LFA-3 micelles occurs when the surfactant is removed or diluted below the critical micelle concentration. The pounding that was done occurs spontaneously. Surfactant removal can be achieved by several techniques. Ru. The main requirement is that the protein is less likely to be adsorbed to other materials (i.e., test tube surfaces, etc.). Rather, the detergent is removed or diluted so that protein aggregation is most favorable. It is to be able to do so. One technique that works well with +nLFA 3 is ultrafiltration and 1 A solution of 96 octyl glucoside in mLFA-3 was concentrated from 2d to 50μl and Diluted with surfactant-free solution. As a result, octyl glucoside diluted below the field micelle concentration (25 nM). For surfactant removal/dilution Other techniques are dialysis and precipitation through detergent-free sucrose. Ta The structure of protein micelles is determined by the nature of the hydrophobic and hydrophilic parts of the protein. Which technique can be used to remove protein micelles, since it does not depend on detergent removal methods. Even if they are generated, they are functionally equivalent.

When is a protein with a hydrophobic component like LFA-3 purified? These proteins may also be stored in glass or plastic containers used during the purification process. It has a tendency to stick. Rapid removal of surfactant counteracts this tendency and protein- plastic.

Protein-glass rather than protein-glass promotes protein-protein binding and results in Approximately 100% of purified LFA-3 can be recovered in the form of micelles. Micelle is PBS Can be maintained for at least 2 months without losing medium activity. Theoretically, if PBS Without proteases, the micelles should have a longer shelf life. Dew.

Micelles created according to the present invention contain hydrophobic lipids or hydrophobic lipids sequestered within the micelles. has a proteinaceous tail” and a spherical hydrophilic region on the outward facing periphery . Figure 3 is a schematic representation of such a micelle containing five monomeric units.

LFA-3 produced by the micellar glue recombinant from the silk recombinant LFA-3 They can also form micelles. Preferred method for producing recombinant LFA-3 is a eukaryotic expression system that enzymatically links PI fans (e.g. Chinese hamster ovary (cHO) cells).

Human TMLFA-3cDNA sequence and deduced amino acid sequence is known (Walluer, B., P., et al., Journal of Experiments). ``Mental Medicine'' current: 923-32 (1987)), which is shown in Ward 4. where potential N-glycosylation sites are indicated by arrows, indicating potential N-glycosylation sites. The lance membrane region is underlined with a dashed line. These array data are E BL, /Reception number from Scene Bank Data Laboratory)'00636 Originally available. The cDNA sequence of the PI type of LFA-3 is different from the TM type. They are identical except they are much shorter. Encodes the PI dagglycan-bound form of LFA-3. The cloned cDNA is expressed in a CHO cell line using standard methods. this These cells recognize the transmembrane region sequence and post-translationally produce the F'I anchor segment. Covalently binds to the rest of the protein. Or TM type of LF A-3 are CHO cells or other cells that cannot bind PI fans, such as mouse L cells. (F Iavell et al. +987).

Recombinant TMLFA-3 or mLFA type B was isolated and, as mentioned above, naturally occurring Micelle formation can occur in the same way as LFA3 present in .

Two MAb saturation concentrations of LFA-3 micelles and CD2 8g AbE Numerous Cs against mLFA-3 binding to T cells or peripheral T cells The potency of D2MAb was tested (Table 1). CD that strongly inhibits E-rosette formation 2MAb was able to completely inhibit LFA-3 binding.

The MAb used in this study is listed in Table 2. 9-1 is Dupo Provided by Dr. nL (Memorial Sloan-Gettering Cancer Institute, NY) , 9.6 was provided by Dr. John Hansen. Song Cancer Research Center, Seattle, WA) 4 I-F to CD2 A-3 Mrs. When 15% bovine serum albumin is adsorbed to 15% bovine serum albumin. , that iodinated LFA-3 binds to CD2-positive cells, and that this binding 2 MAb and LFA-3 MAb have been shown to be inhibited (Du stin et al., supra). According to the present invention, mLFA 3 micelles are Efficient binding to leukemic cells or resting PBL, -T was observed (Table 3 ). Ill I-F A 3 micelle association is TS2. /+8MAb, LFA-3M Figure 5 was inhibited by Ab and unlabeled III-F, A-3 micelles. It is a graph of the time change of dissociation of mLFA-3 from Jurkat cells. . , cells to which mLFA 3 micelles are bound are incubated at 4°C + without nLFA 3. When resuspended in a large volume of medium, the dissociation of +nLFA 3m7 is very It was slow (tl/2″:=-,600 minutes; white blank square), while 10t, tg, / zh7) The dissociation rate of Teha (black squares) was faster in the presence of TS2/l5MAl]. (tl/2=60 minutes). 100μg, /xlTs2/18MAI) When cells are resuspended in soil (black triangles), tl/2 is less than 2 minutes and within 1 hour. Don't completely dissociate. TS2/IsMAb and +nLFA3 are strict competitors to CD2. The rate of dissociation increases in the presence of CD2 MAb as it is thought to bind in a conjugative manner. The fact is that +nLFA-3 micelles bind to cell surface CD2 through multivalent bonds. It shows that there is.

When the micelles come into contact with the surface of the cell, the binding regions of these proteins in the micelles is involved in many interactions, thereby inhibiting the binding of a protein to its target. Gradually increase activity.

Single protein-target interactions tend to dissociate rapidly, whereas micelles In this case, approximately 5 to 10 interactions can occur simultaneously. Hence, the total dissociation The constant is greatly reduced for the micellar form of adhesive proteins due to this multivalent binding. 9 To prove this, we tested LFA 3 micelles against cell surface CD2. and non-micellar 5LFA-3. J) 'from cells and red blood cells LFA-3 showed different degrees of glycosylation. Hence the difference in glycosylation In order to exclude differences in binding caused by Both L7FA-3 and L7FA-3 were isolated. +nLF from JY cells A-3 micelles are similar in size to 1ILFA-3 micelles from red blood cells. , indicates that it is a mixed glue of PI and TM type of FA-3 (Dus tin et al. Nature, above sentence &>, sL FA-3 is +6T, FA-3 Iodinated to the same specific activity, incubated with leukemic T cells for 90 min at 4°C. After incubation, it was spun through an oil cushion. JYmLFA-3 The degree of micellar binding was observed to be 150 times higher than that of JYsLF, A-3 binding. (Table 4).

Consistent with this, unstandardized 1sLFA-3 has a 100-fold excess in weight. However, the binding of mLFA3 to Jurkat cells is not significantly inhibited.

Therefore, multivalent binding contributes to high avidity and LFA3 binding to cell surface CD2. The kidneys are an important factor! Ill.

Complete reversal of mLFA-3 binding by the above G + After 0 minutes, LFA-3 is not endocytosed and during this period This indicates that there is no intramembrane I\ insertion through its PT glycan anchor.

To further test the endocytosis of mLFA-3, LFA-3 was ... Bind to nod cells or PBL at 37°C for 30 minutes or at 4°C for 60 minutes. Ru. Cells were washed with cold medium and then treated with 100 μg/zl TS2,/18 for 6 hours. Incubate with MAb (Table 5). At 4°C, the combined LFA-3 is almost Almost completely released by TS2/18MAI), but at 37°C 50% of Jurkat cells and peripheral blood T lymphocytes ( 20% of PBL-T> is T S 2. /18 Associated with cells even in the presence of MAb (Table 5). TS2. at 37°C. /33 MAb together with mLFA 3 After adding Pj, no association between LFA-3 and cells was detected, and strong association was observed within the plasma membrane. Non-specific lipid-mediated insertion of mL FA-3 into This indicates that it is not a service.

%w+LFA-3 reconstituted into liposomes bound to glass coverslips. The binding to purified CD2 was tested. Iodinated mLFA~3 micelles are It was observed to bind to this solid phase (Table 6). Binding was performed using LFA-3 MAb and TS2. was inhibited by /18M Ab, but not by CD2.1 MAb. Uninhibited, ml-FA3 binds to CD2 without other cell surface components It shows.

Number of +aLFA 3 molecules bound per cell and Jurca-Soto cells and To determine the binding activity of interaction with CD2 on resting and resting PBL-T cells, Binding experiments were performed. The results are shown graphically in Figures 68 and 6b. The percentage of active mLFA 3 protein molecules after coding is 10' By measuring the proportion of bound LFA-3 that can bind to three consecutive aliquots of cut cells. It was decided that This value was between 85 and 95% for different prepared samples.

Specific mLFA3 binding was inhibited by excess T S 2/18 M A b defined as a bond. Binding of chain LFA-3 with control MAb (square) or TS Performed for 1 hour at 4°C in the presence of 2/18 MAb (triangles). MLFA-3 JU Luca Soto Cell (Intention) and PB! , -T (white mark) to be saturated. join to. The Scatchard plot has a Kd of 1 for Jurkat CD2. ．． 7-2.2 nM, while the Kd for P B I--T is 12 16 nM M (Fig. 6b).

This Kd value reflects the extent of binding activity of LFA-3 micelles to each of these cell types. It is. Approximately 420,000 +nLFA-3 monomers as measured at saturation per Soto cell, while PBL-T has approximately 50-80,000 mLF A: Three molecules are connected. These values were measured using ffAb binding to equilibrium. consistent with the relative number of CD2 molecules on Jurkat thin lI& and PB I--T. (Martin, P., et al., Journal Off Immunology - U upper = 180 (19FiO), F' Iunkett, M, L, et al. Journal of Immunology-1364181 (+986)). Non-inhibited CD2. Even if 1 is present at a saturating concentration, both Jurcarito cells) and PBL, -T did not significantly affect binding parameters.

For micelles in a mouthful of −1 tMl, J, FA, 3 micelles alone PBL - Although it has no effect on T proliferation, anti-CD2 MAb is a submitogen. When present at a concentration that acts as a compound, it can induce proliferation of PBL-T. +n　F　A- 3 micelles (40% M) and CD2. IMAb combinations tested in all donors It can act as a powerful mitogen against peripheral blood mononuclear cells (PBMC) from Table 7). Phorbol myristate acetate (PMA) is now available. 4 is required for maximum response, but this response is typically (9 out of 10 donors) ) observed in the absence of exogenous T L-2. m L F, A-3 micelles and and CD2. The proliferation induced by the combination of IM A b is generally caused by phytohemagglutinosis. (PHA), but lower than that obtained with pre-LFA-3, CD2.1 and PHA. M A combination increases thymidine uptake by up to twice as much for some donors as PHA alone increased. mLFA 3 and CD2. IMAb combinations also include nylon wool. (Table 8) to promote division of enriched T cells.

5LFA-3 and CD2. up to a concentration of 800 nM. IMA) + combination <p h, q with or without A) in two experiments for P BMC , two identical donor plants that did not undergo mitogenesis even when sufficient amounts of the substance were present. In the experiment, when CD2.1M Ab was added to mLFA3 (with or without PMA) t2 showed a strong response.

15°, - In the presence of human face cleansing, nylon wool concentrated PBL-T or PMB 9-1 and 9.6/35 for C. Stimulation of IMAb combined monomer proliferation was observed. Table 8). Under these conditions, 9-IMAb and 40% M + nL F A In combination 3, one of the two experiments performed in round-bottomed wells for P There was no effect except in one case, in which case it was increased by exogenous IL-2. I wanted to see a relatively weak stimulus, so the stimulation index was -16.8, whereas the MAb was 11 +9.6. 110). For nylon wool enriched T cells on flat bottom wells, 1 nMP y , q Only in the presence of A1. - FA-3 cooperates with 9 IMAb.

Under certain conditions, mLFA3 cooperates with 9-1λ1Ab for resting T cell activation. In a similar manner, this activation is caused by +oLFA-3 and CD2. I Very weak compared to what is achieved with MAb combinations.

(:D2. Response with rrlLFA-3 with IMAb was accompanied by mLFA-3 For the former, it was stronger and more reproducible than the response of 9-IMAb. Further characterization was performed. Figures 7a and 7b are LFA-3 and CD2.1 Figure 2 is a graph showing the time course and dose response of MAb-induced proliferation. P　B　 MC　p　p　p　-3(40nM)+CD2.1(10μy/z1)(white blank square); +*LFA-3, CD2.1 and IL-2 (black square); or m Started from day 0 to day 3 with LFA 3°CD2.1 and PMA (white circle) The cells were processed for 16 hours until pulse labeling. mLFA-3 and CD2. IM　A　b The stimulation of DNA synthesis by +, :m reached its maximum from day 3 to day 4 (Fig. 7a). . The optimal periods in the presence of exogenously added IL-2 and 1 nM PMA are each It was the 4th day from the 3rd day and the 5th day from the 4th day. +nLFA-3 and CD2.1 MAb dose-responsiveness depends on saturation concentration of other reagents. The decision was made during the current period. mLFA-3 at a concentration showing P B MC without additives (white square), or PMA (1 nM) (black square), D2.1 (10 μg /z12>, or CD2.1 and PMA (white circles). We The cells were pulse labeled for 16 hours on the third day. Saturation CD2.1 MAb (67nM) In the presence of 4 nM mLFA, maximum proliferation of PBMCs or PBLT -3 (Fig. 7b), which shows that mLFA-3 bound to 12 nM PBL-T. The Kd is in the range of 3. Although the presence of exogenous IL-2 did not alter the dose response, The addition of lnMPMA decreased the mT, FA3 concentration for the maximal response by 10 times. I set it. Similarly, in the presence of 40 nM +LFA-3, it gives a maximal response. CD2.1 MAb concentration is 6.7 nM, but in the presence of 1 nMPMA it is only 0 , 67nM was required.

LFA-3”- Special table 13-500659 (5) ,. Proliferative responses to LFA-3 and CD2.1 MAI) Completely blocked by both F　A　-3M　A　b and 9.6M　A　b . 9.6 MAb blocks the binding of mLFA-3 to CD2, but generally CD2.1 MAb is not a co-mitogen (Table 9).

+n LF A-3 and Zr response to CD2.1 MAb CD25 (anti-TL-2 receptor p55 chain) MAb was also blocked; This suggests that it is L-2 dependent. m　F　A　3　OJ:BiCD2 ．． The mitogenic response of PBMCs or PBL-T to 1MAb occurs at 16:00. It is accompanied by a collection of cells within the interval. In the absence of CD2.1 MAb, mLFA3 No clusters were observed due to mLFA-3. This cannot be the result of passive aggregation of cells by micelles.

Silk ■・(″Influence of LFA-3 micelles on a+2 mLFA B activity There are several early indicators of T cell activation that are useful in assessing the ability to activate. how many In that study, the combination with CD2 M A 11 was It has been shown that the flow of C'a + 2 can be stimulated. Therefore, Julkat Stone LFA-3 (alone and and CD2. I MAb) was tested. CCa+2. ]i response is It occurs within 1 minute and can be measured in a single cell, so detection is possible using mLFA 3 micelles. Binding to cell surface CD2 may be due to insertion of LFA-3 into the membrane I\ or on different cells. This indicates that activation signals are being emitted without an opportunity to cross-link with CD2. P.B. Precursor, indo-1 acetoxymethyl ester in L-T or Jurkat cells Load the fluorophore Indo-1 by incubating with Relative 1: Ca”)i has different wavelengths (410n+n/ Flow microfluorimetry using the ratio of Indo-1 fluorescence at 480r+n) Determined by Tory.

+n L F A 3 (up to 240 nM) was added to Jurkat cells. No change in ratio occurred within 15 minutes (at 30-60 seconds) (Figure 9).

Similarly, CD 2.1 M A b (67 nM) alone does not affect Ca+21i. (Figure 9). However, ITILFA 3 and CD 2.1 M　A　I) was added at the same time, continuous at the rate of (Ca+2)i A significant increase occurred (Figure 9), similar to that seen with CD3 MAbs (e.g. 0KT3). Figure 8).

Similar results were obtained in P BLT using 120 nM +nLFA-3. Obtained. There was no change in forward angle light scattering, so the details between these experiments There was no evidence of cell-cell interaction.

(“a+: Movement and equilibrium + nLFA 3. Enables direct comparison of combined data For this purpose, cells were incubated with different concentrations of mLFA3 for 30 min at 37°C. equilibrate and then add CD2.1 MAb to initiate ra+2 transfer. (Figure 10). Under these conditions (Ca + 2) an increase close to half of the maximum of i was found at approximately 1.2 nM ITIL F A3, which was determined as above. Jurcut (”: l’)’) / Kd of mLFA-3 connected Very close to 240 nM mLFA-3 and CD2.1f& Addition of ionomycin to the cells causes a further increase in (Ca+2)i and −1 was not saturated with Ca42 in these experiments. Ru.

Therefore, the combination of mLF, A-3 and CD2.1 transports it through CD2. The signal that leads to an increase in (Ca+2)i is the saturation of CD2 by +n L F A -3 It seems to be proportional to the degree of Figure 10b shows 3 minutes in the activation time plot. 1 is a graph showing the histogram of the intercept at: 0.04 nM (solid line), 0.04 nM (solid line); 4nM (dashed line), 1.2nM (dash-dot line), 4nM (dash-dot line) and 40nM (dotted line).

The protein micelles of the present invention have a standard therapeutic use to competitively inhibit the reactivity of specific surface antigens on target molecules. Be able to do it. In particular, LF A-3 micelles have high levels of cell surface CD2 on T lymphocytes. Treatment by binding with strong binding activity and inhibiting the binding of T lymphocytes to other cells It will be more beneficial.

Furthermore, LFA-3 micelles are co-uptaked with CD2, leading to increased release of CD2 from the cell surface. cause disappearance. This irreversible uptake makes LFA-3 micelles therapeutically effective. It can be used to under-regulate CD2 below the Kd of monomeric LFA-3Kd. .

LFA-3 micelles of the invention in a physiologically compatible carrier such as a saline solution are preferred. When properly administered, a therapeutically effective amount of 0.04 to 4 nM LFA-3 in the blood can be obtained. Get the concentration. Thinking depends on blood volume, and clearance of micelles from the bloodstream. 0.5 to 50 it l? ,/kg of micelles/patient/day is intravenously injected into this concentration. Administered to achieve degree. Micelles are CD2 receptors on the surface of T cells. It works by effectively saturating the cells of other T cells, thereby inhibits binding to This inhibition of binding is sustained by the binding of T cells to other cells. autoimmune diseases such as rheumatoid arthritis. disease; allograft rejection: and graft-to-host disease.

Other implementations are within the scope of the following claims.

Table 1 Signs of inhibition of 3LFA-3 binding by CD 2 M A b Dotorero≧FA-3 Tsutomu ■ (CP M) Control MA b 20,531 ±5 969.6 173 ± 17 TS 2. , /18 153 ± 579-2 154 ± 8 CD2.8 173 ± 29 35.1 7,171 ± 98 CD2.9 164±50 CD 2,1 22,817 ±3629-1 24,092 ±878 D66 16.073 ± 72 0 K T3 21.342 ±487 Binding to Jurkat cells at 4°0 It was held for 1 hour. The input count was 100.000/10' cells. livelihood and a separated material were separated by centrifugation through 15% BSA solution. .

All MAbs are against CD2 except control and OKT3. Conclusion Results are the average of four determinations and are shown with standard deviation and are representative of two experiments. It is something to do.

Mushiji [ TS2/Is CD2 Mouse IEGI Purified 19-I CD2 Mouse l8G3 Purified 3CD2.1 CD2 Mouse IEGI Purified or Ascites 2 CD2.8 CD2 Rat IgG2a Ascites 2CD2.9 CD2 Rat IgG2a Ascites 29.6 CD2 Mouse IgG2a Ascites 4D6 6 CD2 Mouse IgG2b Ascites 59.2 CD2 Mouse IgM Ascites 635.1 C’D2 Mouse IgG2a Ascites 7TS1718L FA-IB Mouse IgGI Jf! Water 1T S 2. /9 L, F A -3 Mouse IEGI Purified 10 K T 3 CD 3 Mouse IgG2 a Culture supernatant 8], Sanchez-Madrid, F., et al., Procedure Day National Academy of Science USA, Reciprocal 4S9 (198 2).

2, ○1ive, D. et al., European Journal of Immunology.

4, K amoun M, et al. Journal of Experimental Medicine N.41U3: 207 (1981).

5, B ernard A, et al. Journal of Experimental Medicine To: 1317 (1,982).

6, B ernard A, et al. (-HD2 research meeting summary: ■Representing the type Leukocytes (A, S, edited by McMichael 1, Oxford University Press, O. +1106<1987)).

7, Martin P, J, et al., Journal of Immunology, 131:180 (1983).

8, Chan H, T, W, et al. Proceedings of the National Academy/L Academy - of Science USA, Japan: 1805 (1981).

L-e 1251-111LF8-3+17 to Jurca Soto cells and resting T cells: ) Combined +251m1. FA-3710' cell (CPM) quadrillion Meiori Kiki Eran 1 Resting T cell control H to b 82,513 33.262+ 111. FA-34,4141,152TS2/Is CD2 MAb 440 93TS2/'9 LFA-3MAb 510 192 binding at 4°C for 1 hour It was held for a while. The amount of 125I-mL F A-3 added was 200. OOOcpm Met. Ties and free radioactivity were determined by centrifugation through a 15% BSA cushion. Separated by Contrast T! ? G1. , T S2/18 and TS2/9 was added at 10Tg/zl, and unlabeled +nLF'A3 was added at 5μ277d. was added in. Results are representative of three experiments with an average of two.

topsoil 1'1 Binding addition of PLC-released LFA-3 to Jurkat n 5LFA 3 + nLFA-3 control MAb 5,553±387 148.760±4003T S2. /18 4,030± 145,023± 23 diurcant cells (10 ’) is 5LFA-3 or +nLFA 3( Both are ryM! prepared from A cells) for 2 hours at 4°C, A portion was centrifuged through an oil cushion.

Accessibility of bound mLFA-3 to extracellular TS2/18 4°C control Control 11,44 1±500 1.543±225TS2/18 Control 86±5 95+14 Control TS2/18 299th26 111±1337℃ Control Control 10, 990±941 1.326±867S2. /is Control 67±1289±1 9 Control TS2/18 4,975i400 390±43 Jyulka Soto cells and P B L-T (5XIO') is 1257-mL F A-3 (100, 0OOCP　M　) and incubated at 4°C for 60 minutes or at 37°C for 30 minutes. Ta. Jurkat cells and PBL-TCD2 are initially 10% occupied by LFA-3. was possessed. Post-incubation at 4°C (with or without TS2/18) ) was carried out for 6 hours to ensure complete dissociation of accessible LFA-3.

Data are the average of four determinations and standard deviations are also shown.

gunwale Control MAb 8,040 TS2/18 MAb 3” CD2.1 MAb 8・122 TS2.9 MAb 612 The number of counts entered was 50,000. 15 minutes or more for binding and free substances It was washed 5 times and separated. The results are the average of two experiments, and the results obtained from three experiments are It represents.

Shishido L Induction buffer for T cell proliferation with mLFA 3 and CD2.1 593 392 417 Teacher 1. F8-3411645549 CD2.1 437 839 646 IL-21,4512,1451,034tn L F^-3+ IL-21, 3451,3991,284CD2.1 + IL-21,6092,0391 ,545PH^　1,713　1.382　834mLTo LF8-3+P^　1. 549 978 828CD2.1+PM^ 1.704 1.252 1.0 09tnL, F^-3-1-CD2. ] 22,294 35,439 47. 478m1, F^-3+CD2.1+IL-222,49737,06545, 384 Kan LF8-3 + CD2.1 + PH^ 77.784 114,313 1 53.240 additives were added on day 0 as indicated: rTL-2 (0,1 021, UF), PMA (1 nM), CD2.1 (1:500 dilution of ascites or or 10ttgy’zl> and tnl-F　A　3　(40 nM). PHA( 1ttg/wR') was approximately 80,000 on days 3 and 4 in these experiments. -100. gave OOOcpm. Wells were treated with 16 hours [3H]-chimidji on the third day. pulse-labeled. Results are the average of three replicates and are representative of 14 experiments. is, La Induction buffer for T cell proliferation with mLFA 3 and 9.1 2.029 262 CD 2.1 3.066 565 CD2.1-1-1-P 4,748 1.1599-1+P M A 10 ．． 520 Write + LFA 3 → -P M A 2.463 2.052 CD2.1 + II ILGA -3263, 32621, 476CD2.1+mT-F A -3+ P M A 330,866 216.9339-1 + T L -2 * 4 , 0789-1 Tenron LFA-3 * 7, 193 1.0749-1 + 蹟LFA-3 +PMA 77.5829-1-1-I#LFA 3+I L-2*34, 0749-1+9.6 *181,570 40.764PB84C and Nye Ronwool enriched T cells are from different donors. P B M C experiment was performed in the presence of 15% heat-inactivated human serum. Those with (4) have round bottoms. It was held in a well.

The 9 additives that produced lower stimulation under corresponding conditions in flat-bottomed wells In addition to day 0, wells were pulse labeled for 16 hours on day 3. 9-IMAb is The 9.6 MAb was added at 1 μg/we: 1:]000 as an ascites dilution. added. Other concentrations are as shown in FIG. The results are the average values of three determinations. The results are representative of two experiments.

Table 9 Inhibition of proliferation by MAb against L, FA-3, CD2 and LFA~1 Harm Control M　A　b　35,439　] 14.313T　S2. ,,’9　1, 684 1.1169.6*776 6.375 PBMC was used. All additives were added on day 0 and wells were On day 1, pulse labeling was performed for 16 hours. For this donor, '9.6+CD2.1 is 549cl)+n and 9.6+CD2.1+PMA is 1663cpta gave. TS2/9MAb is IO, tzy/z? :’Added. 9.6+1 All results are from one experiment. Average of 2 determinations and representative of 3 experiments.

Figure 1 )ζ, exit prong (m ot) 蔚閏(亦) O Engraving (content; no changes) Figure 6-A Figure 6-B Suzumoto+pM) Figure 7-A Nors Jisai〉day Field 7-8 mLFA-3 (nM) Figure 8 mLFA-3CD2.1 Both international search report ゛"9'"0"°0°"°'""P"prTIU5 89 no 018 M

Claims (12)

[Claims] 1. adhesive proteins that naturally contain phosphatidylinositol lipid anchors. micelles, said micelles capable of binding multivalently to multiple target molecules on the cell surface. thing. 2. The micelle of claim 1 comprising less than about 10 molecules of said protein. 3. has binding activity towards cells carrying CD2, and therefore The Kd of LFA-3 is lower than the Kd of monomeric LFA-3 in multimeric and purified LFA-3. . 4. The micelle according to claim 1, wherein the adhesive protein is mLFA-3. . 5. Claim 1, wherein the adhesive protein is synthesized by recombinant DNA technology. Micelles as described in section. 6. A method of inhibiting adhesion of a first cell to a second cell, the method comprising: The cell can bind to molecules on its surface that are present in many types on the second cell, and and proteins that naturally contain phosphatidylinositol lipid anchors, said second cell and said number of molecules on said second cell comprises less than about 10 molecules; contacting a micelle of said protein capable of binding in a multivalent manner with molecules of Method. 7. Method for the treatment of a patient with a medical condition characterized by the presence of an excess of activated T cells administering to the patient LFA-3 micelles in a physiologically relevant buffer; inhibiting binding of the activated T cells in the patient to other cells in the patient; A method of achieving a bloodstream concentration of FA-3 as described above that is effective for. 8. The bloodstream concentration of LFA-3 micelles after administration is 0.04 to 4 nM. The method described in scope item 7. 9. adhesive proteins that naturally contain phosphatidylinositol lipid anchors. A method for increasing binding activity and decreasing dissociation constant, the method comprising: is solubilized in a surfactant solution, and the protein in the surfactant solution is concentrated. , and removing substantially all the surfactant from said solution and sequestering therein. to form adhesive protein micelles with hydrophobic protein ends. How to characterize it. 10. 10. The method of claim 9, wherein said protein is LFA-3. 11. Claim 9, wherein said protein is produced by recombinant DNA technology. How to put it on. 12. A method that affects the activation or proliferation of peripheral blood mononuclear cells or T cells. Therefore, the cells are combined with an antibody against CD2 and contacted with FA-3 micelles. A method characterized by: