JP2706230B2 - Immunosorbent using monoclonal antibody - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to immunoadsorption using a monoclonal antibody which specifically binds to a T cell-replacing factor (hereinafter referred to as TRF). TRF (named IL-5)
It is useful as an immunoadsorbent for use in the purification of. [0002] Specific uses of the immunoadsorbent include quantitative measurement of TRF in a body fluid and purification of TRF. Measurement of TRF in body fluids is essential for the diagnosis of autoimmune diseases due to TRF hypersecretion and immunodeficiency due to poor TRF secretion. The TRF purified by the monoclonal antibody of the present invention is applied to the treatment of immunodeficiency due to poor secretion of TRF and the treatment of autoimmune diseases induced by the production of anti-TRF antibodies in the patient's own body. [0003] The use of the monoclonal antibodies of the present invention and purified TRF could elucidate an unresolved network of immune mechanisms. [0004] Immature B cells are activated by cross-linking antigen receptors with an anti-IgM antibody. TRF binds to the activated receptor on the surface of B cells and promotes B cell proliferation and differentiation into immunoglobulin secreting cells. (Howar
d, M., Nakanishi, K. & Paul, WE, Immunol.Rev.78,185-21
0 (1984) .Kishimoto, T., Ann.Rev., Immunol., 3,133-157
(1985) .Dutton, RW, Folkoff, R., Hirst, JA et al., P.
rog. Immunol., 1,355-386 (1971). Swain, LL, Howard,
M., Kappler. JW, et al., J. Exp. Med., 158, 822-835 (198
3).) TRF is a factor secreted by T cells, and also promotes differentiation of mature B cells that produce antibodies. (Schimpl, A. And
E. Wecker., Nature, 237, NB, 15-17 (1972) .Takatsu, K., T
ominaga, A. & Hamaoka, T., J.Immunol., 124,2414-2422 (1
980) .Takatsu, K., Tanaka, K., Tominaga, A. et al., J. Im
munol., 125, 2646-2653 (1980) .Nakanishi, K. et al., J.
Immunol., 130, 2219-2224 (1983).) Murine T cell fusion cell B151K12 (Takatsu, K. et.
al., J. Immunol., 125, 2646-2653 (1980).) The murine TRF has the following two features. (1)
Induction of IgM secretion and dinitrophenyl (DNP) − by BCL ₁ leukemia B cell line (obtained from the University of Texas)
Induction of IgG class anti-DNP antibody production response in vitro by B cells primed with ovalbumin (OA) complex. (Takatsu, K., Tanaka, K., Tominaga, A. Et
al., J.Immunol., 125,2646-2653 (1980) .Takatsu, K., Har
ada, N., Hara, T. et al., J. Immunol., 134, 382-389 (198
5) .Harada, N., Kikuchi, T., Tominaga, A. et al., J. Immun
ol., 134,3944-3951 (1985). See) (2) BCL ₁ leukemic B cell line proliferation activity. (1) TRF activity, (2) B cell growth factor type II (BCG
Although it may be distinguished from FII) activity in the present specification, since TRF has the activities shown in the above (1) and (2), the activities of the above (1) and (2) are collectively referred to as TRF Let's call it activity. Rat TRF has a molecular weight in the absence of a reducing agent
45,000-60,000 (on gel filtration). Isoelectric point (pI value)
4.7-4.9, B cell stimulating factor 1 (BSF-1), interleukin 2 (IL-2), interleukin 3 (I
L-3), which was different from the immune interferon activity. (Takatsu, K., Harada, N., Hara, T. et al., J. Immu
nol., 134,382-389 (1985) .Harada, N., Kikuchi, T., Tomin
aga, A. et al., J. Immunol., 134, 3944-3951 (1985)) It has recently been proposed to refer to TRF as interleukin 5 (IL-5). However, in this specification, it is unified as TRF. [0005] In the prior art, there was no anti-TRF monoclonal antibody.
Was difficult to purify. In addition, TR of autoimmune disease patients
The blood concentration of F could not be measured. The present invention has been made in view of such problems, and has as its object to provide an immunoadsorbent for TRF. [0006] The immunoadsorbent using the monoclonal antibody of the present invention specifically binds to the antigen portion of the TRF, but it binds to IL-1, IL-2 and IL-3. And B
It comprises a monoclonal antibody that does not bind to SF-1 and a support, and the monoclonal antibody is fixed to the support. As an example of the support, a column for flowing a sample solution can be used. An embodiment of the present invention will be described. Example 1 Preparation of TRF T cell hybridoma B151K12 cell line (Taka
tsu, K. et at., J. Immunol., 125 , 2646-2653 (1980)
PMA (4 β-phorbol 12 β-myristate 12
α-acetate (manufactured by Sigma) was serum-free-cultured in 3 ng / ml for 48 hours to prepare 225 l of a culture supernatant containing TRF. Next, ammonium sulfate fractionation, anion exchange chromatography (DEAE-cellulose), blue sepharose column chromatography, mono-P column (pharma
cia) chromatograph, purified T whose specific activity increased by 1.4 million times by gel chromatography by reverse phase HPLC (manufactured by LKB) using protein C4 (manufactured by Vydac).
RF was obtained. Incidentally, TRF activity was measured by IgM plaque forming cells test using BCL ₁ cells (hereinafter referred to as PFC test.). [0008] The IgM PFC test was performed in the following procedure. Cultured BCL ₁ cell suspension 50 to 100 [mu] l protein A-conjugated sheep red blood cells 10% suspension 50μl agarose 0.5% solution 400μl containing MEM medium were mixed at 45 ° C.,
It was poured on a slide glass and left at room temperature to gel.
Anti-mouse IgM serum was added to the gel and incubated at 37 ° C. for 2 hours and 30 minutes. After adding guinea pig complement and incubating at 37 ° C. for 1 hour, the number of plaques was counted. Anti-D
The NP IgG PFC test was performed in the same manner using 50 to 100 μl of a B cell suspension stimulated with the DNP-OA complex. (B
Since the cells secrete IgG, an operation of adding anti-mouse IgG serum is performed instead of the anti-mouse IgM serum described above. ) T
RF activity was measured using half-maximal respo when purified TRF was used.
The amount giving nses was expressed as a unit (U / culture or PFC / culture) as one unit (U). Preparation of spleen cells Purified TRF emulsified in complete Freund's adjuvant
Wistar rats were immunized with 3.5 × 10 ⁵ U. Wistar rats were further purified with 2 × 10 ⁵ U of purified TRF every 2 days for 20 days.
Boosted. Three days after the final immunization, spleen cells were removed from Wistar rats, and spleen cells were prepared by the following procedure. The spleen was placed in 5 ml of Eagle's MEM culture. Next, 4 ml of the above culture solution was taken in a 10 ml syringe and injected into the spleen. Splenocytes were loosened using forceps. Thereafter, the cell suspension was placed in the syringe and taken out of the syringe three times to reduce the cell mass. The singulated cells were taken out, centrifuged at 1000 to 1200 rpm, and washed. The washed cells were suspended in 2 ml of Eagle's MEM culture solution. Cell fusion Rat spleen cells and mouse myeloma cells P3X63-Ag8.653 (ATCC accession number CRL-
1580) and Koehler and Milstein's cell fusion technology. [0010] After mixing spleen cells 6 × 10 ³ cells with mouse myeloma cells P3X63-Ag8.653 ³ × 10 3 cells to prepare, by centrifugation cells were pelleted. Add 50% PEG- to the pellet.
1 ml of an RPMI-1640 culture solution containing 1500 was gradually added with stirring. Next, the RPMI-1640 culture solution was gradually added and diluted with stirring. Then remove the supernatant by centrifugation and remove 10%
1 × 10 ⁶ cells in RPMI-1640 culture medium containing FCS
1 ml in a 24-well microplate.
Per well. The next day, 500 μl / well HA
T medium was added. After that, repeat the same operation every two days
Cultured for 14 days. Further, the HT medium containing hypoxanthine and thymidine was replaced with the HAT medium at 500 μl / well every two days. 7-10 days later, further change to HT medium and 10% FC
The RPMI-1640 culture medium containing S was exchanged every two days at 500 μl / well. Screening Anti-T of the hybridoma culture supernatant obtained by the above cell fusion
RF activity was determined by TRF activity inhibition and TRF activity absorption tests. TRF activity inhibition test was performed by measuring the IgM PFC test and ³ H-thymidine ⁽³ H-thymidine) uptake. Here, ³ measurements of H-thymidine uptake is, TRF 2U and anti TRF monoclonal antibody to BCL ₁ cells 1.5 × 10 ³ cells cultured in the culture solution 0.2ml containing ³ H-thymidine 0.2μCi the end 6 hours preculture / Wells were added. After washing the cells by centrifugation, ³ H incorporated into the cells was measured by liquid scintillation. In the TRF activity absorption test, the culture supernatant was added to a microplate to which a rabbit anti-rat Ig antibody had been adsorbed and incubated at 37 ° C. for 4 hours. After washing, a certain amount of TRF was added and the mixture was further incubated at 37 ° C. for 4 hours. .
The residual TRF activity of the supernatant was measured. As a result, 61 out of 479 wells containing hybridomas had anti-TR
F activity was exhibited. Further, one well showing reproducible inhibition and absorption activities was selected from the 61 wells. Cloning The hybridoma contained in the selected one well was cloned by the limiting dilution method, and hybridomas TB13 and NC were used.
17 was obtained. Purification of monoclonal antibody An anti-TRF monoclonal antibody was obtained from hybridomas TB13 and NC17 by the following procedure. [0012] The culture supernatant of the hybridoma TB13 was applied to an affinity column using agarose conjugated with goat anti-rat IgG antibody. The adsorbed fraction was eluted with a borate buffer pH = 8.0 containing 3M KSCN to purify the anti-TRF monoclonal antibody TB13. In addition, mass purification was performed by the following operation. After injecting 1 × 10 ⁷ hybridomas TB13 into the intraperitoneal cavity of BALB / C nu / nu mice, about 15 to 20 days later, ascites was collected, and an IgG fraction was obtained by gel filtration using reverse phase HPLC to obtain an anti-TRF monoclonal antibody. Antibody TB13 was purified. The anti-TRF monoclonal antibody NC17 was purified in the same manner using hybridoma NC17. Example 2 (Physicochemical and immunological properties of monoclonal antibodies TB13 and NC17) Identification of immunoglobulin class The immunoglobulin class of anti-TRF monoclonal antibodies TB13 and NC17 was determined by the Ouchterlony method. Anti-TR
F monoclonal antibodies TB13 and NC17 are both Ig
It was G _1. Molecules The molecular weights of the anti-TRF monoclonal antibodies TB13 and NC17 are shown in Table 1 below. [Table 1] [0015] Inhibition To examine the specificity of the specific anti-TRF monoclonal antibody, anti-TRF monoclonal antibody TB13, NC17 is TRF, a BSF-1, IL-1, IL-2, the activity of the lymphokine IL-3 I checked whether to do it. Table 2 shows the results. As can be seen from Table 2, TB13, NC
Each of the 17 anti-TRF monoclonal antibodies was BSF-1,
It does not inhibit IL-1, IL-2, IL-3 activities,
Only activity was inhibited. [Table 2] Note: a) BC for TRF (2U / ml)
L ₁ cell response was determined in cell number of 2 days IgM PFC after culture. b) was measured with a radiation uptake of ³ H-thymidine ⁽³ H-thymidine) after 3 days culture. c) The incorporation of ³ H-thymidine was measured by radiation dose after one day of culture. PC61 (obtained from Swiss Cancer Institute) (Lowenthal, JW,
Zubler, R, H., Nabholz, M. & MacDonald, RH, Nature (Lon
don), 315, 669-672 (1985).) is a rat monoclonal antibody against the anti-interleukin 2 receptor (α-IL-2 receptor). 18F10 (obtained from NIH) (Ohara, J. & Paul, WE, Nature (London), 315, 333-
336. (1985).) Is an IgG ₁ class rat monoclonal antibody against BSF-1. WEHI-3
(Obtained from WEHI (Australia)) is a T cell line that produces IL-3. PC61 50 ng / ml
Is a T cell line GY-1 (Takatsu, K., Harada, N., Hara,
T., et al., J. Immunol., 134, 382-389 (1985).) Inhibits the proliferative response to IL-2. 18 in 30,000-fold dilution
F10 inhibits proliferation of resting B cells by BSF-1 in the presence of anti-IgM antibodies. Examination of specificity, IgM secretion of BCL ₁ cells by TRF, BSF-1 + anti-IgM
Stimulation of resting B cells by antibodies (Ohara, J. & Paul, W.
E., Nature (London), 315, 333-336 (1985).), IL-
Thymocyte stimulation by 1 + PHA (Mizel, SBImmuno
I. Rev., 63, 51-72 (1979)), GY-
Proliferation of one cell, T cell line FDCP- by IL-3
1 Irritation (Kikuchi, Y., Kato, R., Sano, Y., Takahashi,
H., et al., J. Immunol., 136, 3553-3560 (1986).)
Was tested to see if various monoclonal antibodies inhibited it. TR obtained from titer T cell hybridoma B151K12
F is to differentiate BCL ₁ cells into IgM secreting cells, because it has the effect of enhancing the growth, IgM using BCL ₁ cells
PFC and growth tests were performed. [0018] Figure 1A, B is a graph showing the TRF inhibitory potency when adding anti TRF monoclonal antibody TB13, NC17 to BCL ₁ cell culture. Exam is in viv
o 1.5 × 10 ⁵ BCL ₁ cells / 0.2 grown in line
ml were incubated with 2 U of TRF in 200 μl of culture containing various concentrations of anti-TRF monoclonal antibody TB13 or NC17 or anti-BSF-1 monoclonal antibody 18F10. FIG. 1A shows reverse IgM PF after 2 days of culture.
The number of IgM-producing cells was determined by the C test. In the figure, symbol ▼
Measurement of the IgMPFC measured in the medium cultured only BCL ₁ cells, ○ is TRF added, △ is TRF + TB1
3, the measured value at TRF + NC17, the measured value at TRF + 18F10 (extracting in vivo cultured ascites), and the filled square represent the measured value at TRF + TB13 (extracted in vivo cultured ascites). In FIG. 1B, 3H thymidine incorporation was measured after 2 days of culture. In the figure, the symbol ▼ is B
Radiation measurement value when cultured CL ₁ cells alone, is ○ T
Measured value at RF addition, Δ represents measured value at TRF + TB13, ● represents measured value at TRF + NC17, □ represents TRF + 1
8F10 (in vivo culture ascites extraction) represents the measured value, respectively. For comparison, a rat IgG ₁ class anti-BSF-1 monoclonal antibody 18F10 was
It had no secretion inhibition (FIG. 1A) and no inhibition of BCL ₁ cell growth (FIG. 1B). In addition, the anti-TRF monoclonal antibodies TB13 and NC17 each had a strong Ig of 40 ng.
Inhibition of M secretion (FIG. 1A) and growth inhibition of BCL ₁ cells
(FIG. 1B). Next, the anti-TRF monoclonal antibody TB1
3, It was examined whether NC17 inhibits the anti-DNP IgG production response induced by TRF. B cells stimulated with the DNP-OA conjugate were tested for anti-DNP IgG PFC results T
Shows 98 PFC / culture with no RF added, purified TRF3
The addition of U / ml showed 638 PFC / culture. Anti-TR
F monoclonal antibody TB13 50ng / ml was added on the first day of culture, and the anti-DNP IgG PFC test result was 126P.
FC / culture, which clearly inhibited the anti-DNP IgG production response. Similar results were obtained with NC17. This indicates that the anti-TRF monoclonal antibodies TB13 and NC17 inhibit TRF activity in experiments using B cells sensitized with DNP. Example 3 (Application example of anti-TRF monoclonal antibody) Application to immunoadsorbent 2.9 mg of a mixture of anti-TRF monoclonal antibodies TB13 and NC17 was applied to bromine cyanide-activated Sepharose CL-4.
B bound to 3 g was used as an immunosorbent. Reverse-phase HPLC (Parmacia) An effluent obtained by flowing 10 ml (sample 1.2 × 10 ⁴ U) of the culture supernatant of the hybridoma cells B151K12 obtained by gel filtration through the affinity column (3 ml packed volume) was collected. Then, 50 ml of 1M Nacl solution, 100 m of PBS
1. The affinity column was washed with 20 ml of water. Thereafter, 20 ml of a 0.8 M acetic acid solution was flowed to elute the TRF bound to the affinity column. The eluate was freeze-dried and redissolved in 0.8 ml of water. Incidentally, TRF activity was measured by IgM PFC test using BCL ₁ cells.
Table 3 shows the measurement results. [Table 3] As apparent from Table 3, when the crudely purified hybridoma B151K12-produced TRF was applied to the affinity column, 138 U of TRF was eluted.
10.8 × 10 ³ U of TRF eluted with the M acetic acid solution. Normal rat Ig was used as a control column in Sepharose CL.
A similar test was conducted using the sample bound to -4B, and as a result, all TRFs were discharged. Application of TRF to elucidation of physicochemical properties Purified B151K12-produced TRF1 by chloramine T method
× 10 ³ U was labeled with ¹²⁵ I. Unbound ¹²⁵ I was removed from ¹²⁵ I-labeled TRF using Sephadex G-25 (Pharmacia). Next, ¹²⁵ I-labeled TRF
The anti-TRF monoclonal antibody TB13 binding CL-4B
Bound to beads. The complex of ¹²⁵ I-labeled TRF on CL-4B beads and anti-TRF monoclonal antibody TB13 is a buffer containing 2% SDS in the presence (+) and absence (-) of 2-mercaptoethanol (hereinafter referred to as 2ME). Caused the ¹²⁵ I-labeled TRF to dissociate. SDS-PAGE was performed on the eluted fractions. ¹²⁵ I label TR
F was detected by autoradiography. FIG. 3A shows the result. In the figure, number 1 is in the absence of 2ME (−2M
The autoradiograph of the fraction from which ¹²⁵ I-labeled TRF was dissociated from the CL-4B beads in E) is shown. Number 2 is -2
ME with anti-TRF monoclonal antibody TB13-bound CL-
4 shows an autoradiograph when normal rat Ig-bound CL-4B beads were used instead of 4B beads. No. 3 shows ¹²⁵ I-labeled TR from CL-4B beads conjugated with anti-TRF monoclonal antibody TB13 in the presence of 2ME (+ 2ME).
The autoradiograph of the fraction from which F was dissociated is shown. No. 4 shows an autoradiograph when using normal rat Ig-bound CL-4B beads at + 2ME. The gel after SDS-PAGE of the fraction eluted from the anti-TRF monoclonal antibody TB13-bound CL-4B beads under -2ME was sliced every 2 mm along the direction of electrophoresis, and the substances contained in each sliced gel Was eluted electrophoretically. The TRF activity of the eluate was measured using IgM.
It was measured by the PFC test. FIG. 3B shows the result. In FIG. 3A, it is apparent from No. 1 and No. 2 shown as a control that the main fraction has a molecular weight of 44,000 to 48,000 under non-reducing conditions.
Met. Molecular weight 44,000-48,0 under reduction from numbers 3 and 4
The main fraction of 00 (under -2ME) has a molecular weight of 23,000-26,000 (+
2RF), the TRF has a molecular weight of 2
It was found to be a dimer of 3,000-26,000 substances. Moreover, in FIG. 3B, a strong TRF activity was observed in a fraction having a molecular weight of 44,000 to 48,000, particularly about 46,000, and the anti-TRF monoclonal antibody TB13 had a molecular weight of 44,000 to 48,0 having TRF activity.
It was proved to specifically bind to the protein No. 00. The test results in FIG. 1 and Table 2 also show the relationship between TRF and several lymphokines. Anti-TRF
50 ng / ml for monoclonal antibodies TB13 and NC17
Despite significantly inhibiting TRF activity in IL-
The activity of each lymphokine of 1, IL-2, IL-3 and BSF-1 is not inhibited even at a concentration of 10 μg / ml. From the above results, the TRF was BSF-1, IL-1, IL-2, I
It was found to be different from L-3 and to act early. This is a previous report showing that the TRF preparation has no interferon activity (Takatsu, K., et al.,
J. Immunol., 134, 382-389 (1985).) Shows that TRF is a unique lymphokine. Example 4 The TRF used in the previous examples was hybridoma B1.
Since it was obtained from the 51K12 cell line, it was examined whether or not the anti-TRF monoclonal antibody binds to recombinant TRF produced by a genetic engineering technique. Recently, we have discovered that cDN having TRF activity
A was successfully isolated. Isolated PSP6K-mTRF2
3 was cut with SalI to obtain a linear plasmid DNA.
Next, mRNA was synthesized with SP6 RNA polymerase. Xenopus oocytes synthesized RNA
( Xenopus oocytes) and incubated at 20 ° C. for 36 hours. Recombinant TRF (rTRF), i.e. generant is translated PSP6K-mTRF23 in oocytes, stimulates the proliferation and differentiation into IgM secreting cells BCL ₁ cells. In order to examine the inhibitory effect of the anti-TRF monoclonal antibody NC17 on the TRF activity of rTRF, 1.5 × 10 ⁵ /0.2 ml of BCL ₁ cells were added to rTRF in the presence and absence of the anti-TRF monoclonal antibody NC17.
Incubated with RF. Two days after the culture, an IgM PFC test using protein A was performed to determine the number of IgM secreting cells. The results are shown in FIG. FIG. 2 shows the anti-TRF monoclonal antibody NC1.
7 is a graph showing the ability of rTRF to inhibit TRF activity.
In the figure, IgM P symbol ▼ is cultured only BCL ₁ cells
FC measured value, ○: measured value with rTRF (1 U / ml) added, ●: measured value with rTRF (3 U / ml) added, □: measured value with rTRF (1 U / ml) + NC17 added, △
Indicates the measured values obtained when rTRF (3 U / ml) + NC17 was added. FIG. 2 clearly shows that the anti-TRF monoclonal antibody NC17 inhibits the TRF activity of rTRF depending on the amount added. Similarly, an anti-DNP IgG PFC test was performed, and the anti-TRF monoclonal antibody NC17 showed rTR
Whether F-induced IgG secretion was inhibited was examined. DN
B cells stimulated with the P-OA complex showed 169 PFC / culture without rTRF and 1003 PFC / culture with 3 U / ml rTRF. When 1 μg / ml of the anti-TRF monoclonal antibody NC17 was added on the first day of culture, 328 PFC / culture was exhibited, and the anti-D
Inhibited NPI IgG secretion. The above results indicate that the anti-TRF monoclonal antibody NC17 specifically binds to recombinant TRF produced by a genetic engineering technique. As mentioned above,
The monoclonal antibodies of the examples were purified from TRF and
It is useful as a probe for elucidating the molecular properties of RF. The immunoadsorbent using the monoclonal antibody of the present invention is useful as a probe for purifying TRF or elucidating the molecular properties of TRF.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B show anti-TRF monoclonal antibody T;
It is a graph which shows the TRF activity inhibitory ability of B13 and NC17. FIG. 2 shows anti-TRF monoclonal antibody NC17.
Is T of recombinant TRF manufactured by genetic engineering technology
It is a graph which shows RF activity inhibitory ability. FIG. 3A is a photograph showing an autoradiograph of ¹²⁵ I-labeled TRF. FIG. 3B shows TRF at each position of the gel after SDS-PAGE corresponding to FIG.
It is a graph which shows activity ability.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12N 15/09 C12P 21/08 C12P 21/00 G01N 33/577 B 21/08 9282-4B C12N 15 / 00 A G01N 33/577 9282-4BC (C12P 21/00 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (1)

(57) [Claims] It specifically binds to the antigen portion of TRF, but IL-
1. An immunoadsorbent comprising a monoclonal antibody that does not bind to IL-2, IL-3 and BSF-1, and a support, wherein the monoclonal antibody is fixed to the support. 2. The immunoadsorbent according to claim 1, wherein the TRF is a cell-derived TRF. 3. The immunoadsorbent according to claim 1, wherein the TRF is a recombinant TRF produced by a genetic recombination technique. 4. The immunoadsorbent according to claim 1, wherein the support is a column for flowing a sample solution.